Sod polypeptide analogs

ABSTRACT

A novel analog of human superoxide dismutase which comprises the non-acetylated, non-glycosylated form of natural human superoxide dismutase having the ser-met amino acid sequence attached to the N-terminus and a mixture comprising this analog and non-acetylated, non-glycosylated superoxide dismutase having an amino acid sequence identical to that of natural human superoxide dismutase are provided. Methods of catalyzing a chemical reaction using the novel analog and mixture are provided.

This application is a divisional of U.S. Ser. No. 449,125, filed Dec. 8,1989, now U.S. Pat. No. 5,162,217; which is a continuation of U.S. Ser.No. 202,238, filed Jun. 3, 1988, now abandoned; which was a continuationof U.S. Ser. No. 897,056, filed Aug. 14, 1986, now abandoned; which wasa continuation-in-part of U.S. Ser. No. 767,143, filed Aug. 19, 1985,now abandoned; which was a continuation-in-part of U.S. Ser. No.644,245, filed Aug. 27, 1984, now abandoned.

BACKGROUND OF THE INVENTION

One aspect of genetic engineering involves the insertion of foreign DNAsequences derived from eucaryotic sources into Escherichia coli or othermicroorganisms. A further refinement of genetic engineering concernsinducing the resulting microorganism to produce polypeptides encoded bythe foreign DNA. Production of polypeptides can be considered a two-stepprocess, with each step including numerous substeps. The two steps aretranscription and translation. To produce a polypeptide efficiently andin quantity both steps of the process must be efficient. Transcriptionis the production of mRNA from the gene (DNA). Translation is theproduction of polypeptide from the mRNA.

A critical substep of the transcription process is initiation, that is,the binding of RNA polymerase to a pro-moter-operator region. Thesequence of deoxyribonucleotide bases which make up the promoter regionmay vary and thereby effect the relative efficiency of the promoter. Theefficiency depends on the affinity of the RNA polymerase for thepromoter.

The efficiency of translation is affected by the stability of the mRNA.Increased stability of the mRNA permits improved translation. Althoughthe exact determinants of mRNA stability are not precisely known, it isknown that mRNA secondary structure as determined by the sequence of itsbases has a role in stability.

The initial substep of translation involves binding of the ribosome to abase sequence on the mRNA known as the Shine-Dalgarno sequence or theribosomal binding site (RBS). The synthesis of polypeptides begins whenthe ribosome migrates along the mRNA to the AUG start codon fortranslation. Generally these codohs are found approximately 10 bases"downstream" from the Shine-Dalgarno site. Factors which increase theefficiency of translation include those which enhance binding of theribosomes to the Shine-Dalgarno site. It has been shown that thestructure of the mRNA in the region of the Shine-Dalgarno sequence andthe AUG codon and the distance between the Shine-Dalgarno sequence andthe AUG codon each play a critical role in determining the efficiency oftranslation. Other factors which affect the efficiency of translationare premature termination and attenuation. Efficiency of translation canbe improved by removing the attenuation sites.

A difficulty encountered in attmpts to produce high amounts ofeucaryotic polypeptides in bacterial cells involves the inability ofcells producing large amounts of mRNA to grow efficiently. Thisdifficulty can be eliminated by preventing transcription by a processknown as repression. In repression genes are switched off due to theaction of a protein inhibitor (repressor protein) which preventstranscription by binding to the operator region. After microorganismshave grown to desired cell densities, the repressed genes are activatedby destruction of the repressor or by addition of molecules known asinducers which overcome the effect of the repressor.

Numerous reports may be found in the literature concerning the cloningof eucaryotic genes in plasmids containing the P_(L) promoter from λbacteriophage. (Bernard, H. V., et al., Gene (1979) 5, 59; Derore, C.,et al., Gene (1982) 17, 45; Gheysen, D., et al., Gene (1982) 17, 55;Hedgpeth, J., et al., Mol. Gert. Genet. (1978) 163, 197; Remaut, E., etal., (1981) Gene 15, 81 and Derynck, R., et al., Nature (1980) 287, 193.In addition, European Patent Application No. 041,767, published Dec. 16,1981, describes expression vectors containing the P_(L) promoter frombacteriophage. However, none of these references describe the use of theC_(II) ribosomal binding site.

The use of a vector containing the P_(L) promoter from λ bacteriophageand the C_(II) ribosomal binding site has been described. (Oppenheim, A.B., et al., J. Mol. Biol. (1982) 158, 327 and Shimatake, H. andRosenberg, M., Nature (1981) 292, 128.) These publications describe theproduction of increased levels of C_(II) protein but do not involve ordescribe the production of eucaryotic proteins.

Other vectors which contain the P_(L) promoter and the C_(II) ribosomalbinding site have also been described (Courntey, M., et al., PNAS (1984)81: 669-673; Lautenberger, J. A., et al., Gene (1983) 23:75-84 andLaurenberger, J. A., et al., Science (1983) 221: 858-860). However, allof these vectors lead to the production of fused proteins which containthe amino terminal portion of the C_(II) protein.

In 1982 Shatzman and Rosenberg presented a poster at the 14th MiamiWinter Symposium (Shatzman, A. R. and Rosenberg, M., 14 Miami WinterSymposium, abstract p98 [1982]). This abstract provides a non-enablingdisclosure of the use of a vector containing P_(L) from λ bacteriophage,Nut and the C_(II) ribosomal binding site to synthesize a "eucaryotic"polypeptide (SV40 small T antigen is actually not a eucaryoticpolypeptide but a viral protein) in an amount greater than 5% of thecell protein in an unnamed bacterial host. The operator used is notdefined. Neither an origin of replication nor a gene for a selectablephenotype is identified. This system with which the vector is used isdescribed as including certain host lysogens into which the vector canbe stably transformed.

Applicants are aware of the existence of a pending U.S. patentapplication in the name of M. Rosenberg filed under Ser. No. 457,352 ,now U.S. Pat. No. 4,578,355, by the National Institutes of Health, Dept.of Health and Human Services, U.S.A. Portions of this application havebeen obtained from the National Technical Information Service, U.S.Dept. of Commerce. However, the claims are not available and aremaintained in confidence. The available portions of the application havebeen reviewed. It indicates that the host is important (p8, line 17) butfails to identify any suitable host. It further depends upon the use ofa λ mutant which is not specified (p4, line 20). It indicates that thehost contains lysogens (p8, line 18) unlike the present invention inwhich the host is not lysogenic. It mentions cloning and expression of aeucaryotic gene, monkey metallothionein gene, (p7, line 18) but does notprovide details. It specifies that neither the sequence nor the positionof any nucleotide in the C_(II) ribosomal binding region has beenaltered (p 3, line 27).

Pending, co-assigned U.S. patent application Ser. No. 514,188, filedJul. 15, 1983, now abandoned, describes novel vectors useful for theexpression of polypeptides in bacteria.

These vectors include λ P_(L) O_(L), the N utilization site for bindingantiterminator N protein, a ribosomal binding site, an ATG codon, arestriction enzyme site for inserting a gene encoding a desiredpolypeptide, an origin of replication and a selectable marker. In thesevectors the distance between the N utilization site and the ribosomalbinding site is greater than about 300 base pairs. In addition, each ofthese vectors contains a specific ribosomal binding site which cannot bereadily replaced. These vectors are not equally useful for expression ofdifferent polypeptides.

U.S. Ser. No. 514,188 now abandoned, also discloses a method ofproducing the polypeptide encoded in the vector by growing a hostcontaining the vector, inducing polypeptide expression and recoveringthe polypeptide.

Superoxide dismutase (SOD) and analogs thereof are some of severalpolypeptides which may be produced using the vector and methodsdisclosed in U.S. Ser. No. 514,188, now abandoned..

The present invention relates to expression plasmids which unexpectedlyprovide enhanced expression of superoxide dismutase and analogs thereof.By employing different ribosomal binding sites in the plasmids of thisinvention it is possible to achieve enhanced expression levels ofsuperoxide dismutase or analog thereof relative to the levels achievedwith the previous vectors. In addition, using the same ribosomal bindingsites as in the previous vectors, it is possible to achieve enhancedexpression of superoxide dismutase or the analog.

The present invention also relates to a method for enhanced productionof SOD and analogs thereof in bacteria, including prototrophic and lyricbacteria, utilizing these plasmids.

The present invention also provides for plasmids and methods whichexclusively produce the non-acetylated analog of human CuZn superoxidedismutase having an amino acid sequence identical to that of naturalhuman CuZn superoxide dismutase.

Superoxide dismutase is of considerable interest because of itspharmacological properties. Bovine-derived, naturally-occurringsuperoxide dismutase (orgotein) has been recognized to possessanti-inflammatory properties and is currently marketed in certainEuropean countries, e.g., West Germany, for use in the treatment ofinflammation. It is also sold in a number of countries including theUnited States as a veterinary product for treating inflammation,particularly for treating inflamed tendons in horses.

Additionally, the scientific literature suggests that SOD may be usefulin a wide range of clinical applications. These include prevention ofoncogenesis and tumor promotion and reduction of cytotoxic andcardiotoxic effects of anti-cancer drugs (Oberley, L. W. and Buettner,G. R., Cancer Research 39, 1141-1149 (1979)); protection of ischemictissues (McCord, J. M. and Roy, R. S., Can. J. Physiol. Pharma. 60,1346-1352 (1982)), and protection of spermatozoa (Alvarez, J. G. andStorey, B. T., Biol. Reprod. 28, 1129-1136(1983)). In addition, there isa great interest in studying the effect of SOD on the aging process(Talmasoff, J. M., Ono, T. and Cutler, R. G., Proc. Natl. Acad. Sci. USA77, 2777-2782 (1980)).

The present invention also relates to using human superoxide dismutaseanalogs to catalyze the reduction of superoxide radicals in the presenceof H⁺, to hydrogen peroxide and molecular oxygen. In particular, thepresent invention concerns using hSOD analogs to reduce reperfusioninjury following ischemia and prolong the survival period of excisedisolated organs. It also concerns the use of hSOD or analogs thereof toreduce injury on reperfusion following organ transplantation and spinalcord ischemia. These analogs may also be used for bronchial pulmonarydysplasia.

The human CuZn superoxide dismutase and analogs thereof of the presentinvention are commercially advantageous in that they are less toxic thanorgotein and have enhanced stability to lyophilization while retainingtheir enzymatic activity.

SUMMARY OF THE INVENTION

A plasmid for the production of superoxide dismutase or analog thereofwhich upon introduction into a suitable bacterial host cell containingthe thermolabile repressor C_(I) renders the host cell capable, uponincreasing the temperature of the host cell to a temperature at whichthe repressor is inactivated of effecting expression of DNA encodingsuperoxide dismutase or the analog and production of superoxidedismutase or the analog comprising:

a double-stranded DNA molecule which includes in 5' to 3' order thefollowing:

a DNA sequence which contains the promoter and operator P_(L) O_(L) fromlambda bacteriophage;

the N utilization site for binding antiterminator N protein produced bythe host cell;

a first restriction enzyme site permitting replacement of the DNAsequence containing the ribosomal binding site which follows thereafter;

a DNA sequence which contains a ribosomal binding site for rendering themRNA of the gene encoding superoxide dismutase or analog thereof capableof binding to ribosomes within the host cell;

an ATG initiation codon;

a second restriction enzyme site;

gene encoding superoxide dismutase or the analog thereof in phase withthe ATG initiation codon;

and which additionally includes a DNA sequence which contains an originof replication from a bacterial plasmid capable of autonomousreplication capable of autonomous replication in the host cell and a DNAsequence which contains a gene associated with a selectable oridentifiable phenotypic trait which is manifested when the plasmid ispresent in the host cell, the distance between the 3' end of the P_(L)O_(L) promoter and operator sequence and the 5' end of the N utilizationsite being less than about 80 base pairs and the distance between the 3'end of the N utilization site and the 5' end of the ribosomal bindingsite being less than about 300 base pairs.

The plasmids of the invention can be introduced into suitable hostswhere the gene for superoxide dismutase or SOD analog can be expressedand the superoxide dismutase or analogs thereof produced. The presentlypreferred plasmids for human superoxide dismutase are: pSOD β₁ T₁₁,pSODβMAX₁₀ and pSODβMAX₁₂. Preferred hosts include Escherichia coli, inparticular, autotrophic E. coli A1645; prototrophic E. coli A4200 andA4255, and lytic E. coli A4048.

A1637 was obtained from C600 by inserting transposon containingtetracycline resistance gene within the galactose operon as well as thelambda system for expression which is close to galactose operon. C600 isavailable from the American Type Culture Collection, as ATCC AccessionNo. 23724.

A1645 was obtained from A1637 by selection for Gal⁺ (ability to fermentgalactose) as well as loss of tetracycline resistance. It still containsthe lambda expression system but part of the transposon has been removedby selection. Its phenotype is C600 r⁻ m³⁰ gal⁺ thr⁻ leu⁻ lacZ⁻ bl(λc1857 ΔH1 ΔBamH1 N⁺).

A1645 is presently a more preferred strain for expression of genesencoding superoxide dismutase or analogs thereof. It has been depositedwith the American Type Culture Collection in Rockville, Md., U.S.A.containing various plasmids as described more fully hereinafter. Alldeposits were made pursuant to the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms except that pBR322 and pBRMare fully available from the American Type Culture Collection as ATCCAccession Nos. 37017 and 37283, respectively, the D4 was deposited underATCC Accession No. 31826 in connection with the filing of a U. S. patentapplication.

Prototrophic strains of Escherichia coli, which enable high levelpolypeptide expression even when grown in a minimal media may also beused as hosts for the vectors of this invention. Preferred prototrophicstrains include A4200 and A4255. Strain A4255 containing the plasmidp9200 has been deposited with the ATCC under Accession No. 53215. Evenmore preferred are biotin independent prototrophic strains such as A4346containing the plasmid pGH44 which has been deposited with the ATCCunder Accession No. 53218.

Lytic strains of Escherichia coli may also be used as hosts for thevectors of this invention. Suitable lyric strains include those whichproduce, at the temperature at which the polypeptide is produced but ata rate slower than that at which the polypeptide is produced, asubstance e.g., an enzyme like endolysin which will cause the cell tolyse.

This permits the cell to produce relatively large amounts of the desiredpolypeptide before the amount of the lysing substance produced reachesthe level which causes cell lysis. Examples of suitable lytic strainsinclude those containing PlcI^(ts) plasmid such as strain A4048containing pGH44 which has been deposited with the ATCC under AccessionNo. 53217.

The resulting host vector systems can be employed to manufacturesuperoxide dismutase or superoxide dismutase analogs. Host cellscontaining the plasmids are grown under suitable conditions permittingproduction of superoxide dismutase or the analog and the resultingsuperoxide dismutase or analog is recovered. Using the host vectorsystems, analogs of human superoxide dismutase have been prepared.Veterinary and pharmaceutical compositions containing these SOD analogsand suitable carriers have also been prepared. These superoxidedismutase analogs have been used to catalyze the following reaction:

    2O.sub.2.sup.- +2H.sup.+ →H.sub.2 O.sub.2 +O.sub.2

More particularly, these analogs have been used to reduce injury causedby reperfusion following ischemia or organ transplantation, reducecardiac infarct size, increase the survival time of excised isolatedorgans, and reduce spinal cord in jury.

DESCRIPTION OF THE FIGURES

The restriction maps for each of the plasmids shown in FIGS. 1-31 do notidentify all restriction sites present on each plasmid. In some casesrestriction sites are shown in one figure but not in another. However,in all cases those restriction sites necessary for a completeunderstanding of the invention are shown.

FIG. 1. Construction of pAL500.

A plasmid containing bGH cDNA, D4 (ATCC No. 31826), was digested withHaeII. The resulting 1600 base pair large fragment was purified anddigested at 37° C. for 5 minutes with S1 exonuclease. A synthetic EcORIlinker with the sequence: ##STR1## was attached to the ends of theresulting fragments by ligation, The ligation mixture was cleaved withEcoRI and inserted into pBR322 (ATCC No. 37017) which had been cleavedwith EcoRI. A clone, pALRI, was obtained which upon cleavage with EcoRIreleased a 1200 base pair fragment with the sequence: ##STR2## at the 5'end. This sequence demonstrates that pALRI contains an EcoRI restrictionsite which includes the TTC codon for residue number 1 (phenylalanine)of natural bGH. pALRI was subjected to a partial cleavage with PstI. Thedigest was treated with DNA polymerase I large fragment (Klenow) andHindIII linkers with the sequence: ##STR3## were attached by ligation.The ligation mixture was cleaved with EcoRI and HindIII. The fragmentcontaining bGH cDNA was isolated and subcloned into pBR322 between theEcoRI and HindIII restriction sites to give pAL500 (ATCC No. 39782).

FIG. 2. Construction of pRO211 and pRO12.

The plasmid pJH200 (ATCC No. 39783) was partially digested with NdeI,treated with DNA polymerase I (Klenow) to fill in the ends and theresulting ends were religated to form the expression vector pRO211. Theexpression vector pRO211 was digested with NdeI and HindIII, the largefragment isolated and ligated to an NdeI-HindIII bGH fragment isolatedfrom pAL500 (ATCC No. 39782) to give pRO12. (The NdeI-HindIII fragmentwas produced from pAL500 by digesting it with EcoRI and ligating to theends of the digestion product synthetic linkers with the sequence:##STR4##

The ligation mixture was digested with NdeI and HindIII and theresulting NdeI-HindIII bGH fragment isolated.)

FIG. 3. Construction of pSAL 5200-6

pRO12 (FIG. 2) was partially digested with PvuII followed by digestionwith NdeI to eliminate a 72 base pair fragment. A synthetic DNA fragmentcoding for the first 24 amino acids of the N-terminus of authentic bGHwas ligated to the digested pRO12.

The synthetic DNA fragment was constructed by annealing twophosphorylated synthetic single-stranded DNAs of the sequence: ##STR5##

The annealed fragment was treated with DNA polymerase I (Klenow) in thepresence of all four deoxyribonucleoside triphosphates in order to formthe full length double-stranded DNA. The fragment was digested withPvuII and NdeI before ligation to pRO12 to form pSAL 5200-6.

FIG. 4. Construction of p3008.

p3008 (ATCC No. 39804) was constructed by ligating NdeI-digested pRO211(FIG. 2) with the pGH fragment isolated from an NdeI digest of theplasmid ppGH-NdeI/RI.

ppGH-NdeI/RI contains full length pGH cDNA to both ends of which NdeIsites have been added by means of synthetic linkers.

FIG. 5. Construction of p5002.

p5002 was constructed by tripartite ligation of a dimerized syntheticlinker and the 2 cGH fragments isolated from an NdeI and BanII digest ofthe plasmid pcGH-NdeI/RI. The ligation mixture was digested with NdeIand then ligated to the expression vector pRO211 (FIG. 2) after it hadrestricted with NdeI. A colony containing the plasmid p5002 wasisolated.

The synthetic linker was constructed from two single-stranded syntheticDNAs of the sequence: ##STR6##

The linker was phosphorylated before ligation. The linker codes for thefirst 18 amino acids of the N-terminus of the authentic cGH.

The plasmid pcGH-NdeI/RI contains full length cGH cDNA at the 5' end ofwhich there is an EcoRI restriction site and at the 3' end of whichthere is an NdeI restriction site. These restriction sites were added bymeans of synthetic linkers.

FIG. 6. Construction of pHG44 and pHG50.

pRO12 (FIG. 2) was digested with HindIII. The linear form DNA (form III)was purified from agarose gel and ligated to a HindIII-HindIII fragmentof about 1200 base pairs which contains the rRNA operon transcriptiontermination sequences T₁ T₂. The T₁ T₂ HindIII-HindIII fragment wasisolated from plasmid pPSl (ATCC No. 39807) which had been digested withHindIII. The resulting plasmid pHG44 (ATCC No. 39806) contains the T₁ T₂sequences at the 3' end of the recombinant (rec) bGH sequence.

The plasmid pSK434 (ATCC No. 39784) containing the λcI⁴³⁴ repressorsequences was digested with HpaII. The λcI⁴³⁴ HpaII-HpaII fragment wasisolated and ligated to pHG44 which had been digested with ClAI. Theresulting plasmid pHG50 (ATCC No. 39805) contains the T₁ T₂transcription termination sequences and the λcI⁴³⁴ repressor sequence.

FIG. 7. Construction of p8300-10.

The plasmid p8300-10A (ATCC No. 39785) which expresses an analog of thenatural phenylalanine form of bGH having methionine at the N-terminus(met-phe bGH) was prepared as follows. The plasmid p7200-22 contains theλP_(L) promoter and ribosomal binding site derived from pJH200 (ATCC No.39783), DNA encoding met-phe bGH and the T₁ T₂ rRNA terminationsequences. The ClaI-ClaI fragment containing the λP_(L) promoter, theC_(II) ribosomal binding site, the met-phe bGH gene and the T₁ T₂transcription termination sequences was inserted into the unique ClaIsite of plasmid pOPlΔ6, a constitutive high copy number plasmid, to formp8300-10A.

FIG. 8. Construction of pSAL-130/5 and pSAL-170/10.

The plasmid pHG44 (ATCC No. 39806) expressing met-asp-gln bGH proteinwas digested with NdeI and HindIII. The resulting NdeI-HindIII bGHfragment was isolated and ligated to a fragment from p8300-10A (ATCC No.39785) prepared by partial digestion with both NdeI and HindIII. Such aligation replaces the met-phe bGH gene fragment with the met-asp-gln bGHgene fragment. The plasmid so obtained, pSAL-130/5, expresses rec bGH.pSAL-170/10 was obtained by treating the EcoRI-AvaI fragment containingthe Tet^(R) gene of pBR322 plasmid (ATCC No. 37017) with DNA polymeraseI (Klenow) and inserting it into pSAL-130/5 which had been digested withBamHI and filled in with DNA polymerase I (Klenow).

FIG. 9. Construction of pSAL-210/4.

Linear form DNA (form III) was prepared by partial ClaI digestion ofpSAL-170/10. It was purified from an agarose gel and ligated to aHpaII-HpaII cI⁴³⁴ gene fragment which was isolated from a HpaII digestof the plasmid pSK434 (ATCC No. 39784).

FIG. 10. Construction of pSAL 5600-1.

pSAL 5200-6 (FIG. 3) was digested with HindIII. The linear form DNA(form III) was purified from an agarose gel and ligated to aHindIII-HindIII fragment of about 1200 base pairs which contains therRNA operon transcription termination sequences, T₁ T₂. The T₁ T₂HindIII-HindIII fragment was isolated from the plasmid pPS1 (ATCC No.39807) which was digested with HindIII. The resulting plasmid pSAL5600-1 contains the T₁ T₂ sequences at the 3' end of the met-asp-gln bGHsequence.

FIG. 11. Construction of p3009.

The NdeI-NdeI pGH fragment was isolated from plasmid p3008 (ATCC No.39804) (FIG. 5). The fragment was inserted into the unique NdeI site ofthe expression vector p579 (FIG. 19) which had been digested with NdeI.The resulting plasmid p3009 expresses an analog of natural porcinegrowth hormone protein having a methionine residue added at theN-terminus.

FIG. 12. Construction of p5003.

The NdeI-NdeI cGH fragment was isolated from plasmid p5002. The fragmentwas inserted into the unique NdeI site of the expression vector p579(FIG. 19) which had been digested with NdeI. The resulting plasmid p5003(ATCC No. 39792) expresses an analog of natural chicken growth hormoneprotein having a methionine residue added at the N-terminus.

FIG. 13. Construction of pSODα2.

The pJH200 (ATCC No. 39783) expression vector was digested with NdeI.The 550 base pair NdeI fragment containing the λP_(L) promoter andC_(II) ribosomal binding site was isolated and inserted into the uniqueNdeI site of plasmid pSOD NH-10 which had been digested with NdeI.(Plasmid pSOD NH-10 is derived from a cDNA clone of human SOD[Lieman-Hurwitz, J., et al., PNAS (1982) 79: 2808]) The resultingplasmid pSOD NH-550 was digested with AluI. (Only the relevant AluI siteis shown in the figure.) The large AluI fragment containing the AP_(L)promoter and the SOD gene was isolated. BamHI linkers were attached andthe resulting fragment was digested with BamHI. The BamHI digestionproduct was inserted into the unique BarnHi site of pBRM (ATCC No.37283) to form pSODα2 (ATCC No. 39786).

FIG. 14 Construction of pSODαl3 and pSOD⊕1.

The plasmid pSODα2 (ATCC No. 39786) was partially digested with EcoRIand the resulting linear form DNA was isolated from an agarose gel. Thepurified DNA was filled in with DNA polymerase I (Klenow) and religated,The resulting clone pSODαl3 contains one EcoRI site located at the 5'end of the ribosomal binding site. A fragment containing the β-lactamasepromoter and ribosomal binding site was isolated from plasmid BLAII(ATCC No. 39788) which had been digested with EcoRI and AluI. The 200base pair fragment was ligated to the large fragment isolated frompSODαl3 which had been digested with NdeI, filled in with DNA polymeraseI (Klenow) and then digested with EcoRI. The resulting plasmid pSODβ1contains the ribosomal binding site of the β-lactamase gene and theλP_(L) promoter.

FIG. 15, Construction of pSODβ₁ T₁₁.

Plasmid pBR322 (ATCC No. 37017) was digested with EcoRI and AvaI. Theresulting DNA was filled in with DNA polymerase I (Klenow), The Tet^(R)gene fragment was then isolated and ligated to the large fragmentisolated from pSODβ1 (FIG. 14) plasmid which had been digested with PstIfollowed by a partial BamHI digest and then filled in with DNApolymerase I (Kienow), The resulting plasmid pSODβ₁ T₁₁ (ATCC AccessionNo. 53468) contains the Tet^(R) gene.

FIG. 16. Construction of pSODβ₁ TT-1.

The rRNA T₁ T₂ transcription termination fragment was isolated fromplasmid pPSl (ATCC No. 39807) which had been digested with HindIII andfilled in with DNA polymerase I (Klenow). The fragment was ligated toplasmid pSODβ₁ T₁₁ (FIG. 15) which had been partially digested withBamHI and filled in with DNA polymerase I (Klenow).

FIG. 17. Construction of pSODβ₁ - BA2.

A synthetic DNA fragment with the sequence: ##STR7## which is similar tothe sequence of the natural β-lactamase ribosomal binding site, wasphosphorylated and ligated to the large fragment of pSOD⊕l3 plasmid(FIG. 14) which had been digested with NdeI and EcoRI.

FIG. 18. Construction of pTV-188.

Plasmid pApoE-EX2 (ATCC No. 39787) was digested with NdeI and thenfragments filled in with DNA polymerase I (Klenow). The resulting ApoEgene fragment was isolated and inserted into the unique blunt end StuIsite of the pSODβ₁ T₁₁ plasmid (FIG. 15). The resulting plasmid pTV-188expresses an ApoE fused protein.

FIG. 19. Construction of p579.

The rRNA operon T₁ T₂ transcription termination fragment was isolatedfrom plasmid pPSl (ATCC No. 39807) which had been digested with HindIII.The T₁ T₂ fragment was inserted into the unique HindIII site of pRO211(FIG. 2) which had been digested with HindIII. The resulting expressionvector, p579, contains the λP_(L) promoter, the C_(II) ribosomal bindingsite, followed by the T₁ T₂ transcription termination signals.

FIG. 20. Construction of pTV-170.

The NdeI-NdeI ApoE fragment was isolated from plasmid pApoE-EX2 (ATCCNo. 39787) and inserted into the unique NdeI site of the expressionvector p579 (FIG. 19) which had been digested with NdeI. The resultingplasmid pTV-170 expresses an analog of natural human ApoE protein havinga methionine residue added at the N-terminus.

FIG. 21. Construction of pTV-190.

The plasmid pTV-170 (FIG. 20) was partially digested with NdeI andfilled in with DNA polymerase I (Klenow). The isolated linear form DNAwas religated to yield the plasmid pTV-190 which was analyzed and foundto have only one NdeI site at the 5' end of the ApoE gene.

FIG. 22. Construction of pTV-194.

The β-lactamase promoter and ribosomal binding site fragment wasisolated from plasmid pBLA11 (ATCC No. 39788) after digestion with EcoRIand AluI. This fragment was ligated to the large fragment of pTV-170(FIG. 20) plasmid which had been digested with NdeI, filled in with DNApolymerase I (Klenow) and then digested with EcoRI.

FIG. 23. Construction of pSAL 160-5.

An AvaI-AvaI fragment containing the ApoE DNA sequence was isolated frompTV-170 (FIG. 21) which was digested with AvaI. The fragment was filledin with DNA polymerase I (Klenow) and isolated on agarose gel. Thepurified ApoE fragment was inserted into the PstI site of the pTV 104(2)plasmid (ATCC No. 39384) which was partially digested with PstI andfilled in with DNA Polymerase I (Klenow). The resulting plasmid isdesignated pSAL 160-5.

FIG. 24. Construction of pTV-214.

A synthetic fragment containing the first 14 amino acids of human growthhormone with the sequence: ##STR8## was phosphorylated using .sup.γ32p-ATP and polynucleotide kinase. The phosphorylated linker was insertedinto the unique NdeI site of pTV-190 plasmid which had been digestedwith NdeI.

FIG. 25. DNA Sequence of the Cloned hCuZnSOD in pSODβ₁ T₁₁

FIG. 25 shows the presence and location of two ATG codons located at the5' end of the cloned human CuZn SCD gene in pS0Dβ₁ T₁₁ (ATCC AccessionNo. 53468) which was constructed as shown in FIG. 15.

FIG. 26. Construction of pΔRB Plasmid

Tet^(R) expression vector, pΔRB, was generated from pSODβ₁ T₁₁ (ATCCAccession No. 53468) by complete digestion with EcoRI followed bypartial cleavage with BamHI restriction enzymes. The digested plasmidwas ligated with synthetic oligomer ##STR9## resulting in pΔRBcontaining the λPL promoter. pΔRS contains unique restriction sites forinsertion of ribosomal binding sites and genes downstream of the P_(L)promoter.

FIG. 27. Construction of pβUN

Construction of pβUN, a general purpose expression vector, containingthe λP_(L) promoter and β-lactamase promoter and RBS is shown in FIG.27. Unique NdeI site followed by SmaI, XbaI and BgII sites wereintroduced downstream of the β-lactamase RBS for insertion of anydesired gene. It should be pointed out that 3 nucleotide changes weremade at the 3+ end of the β-lactamase RBS-, one to eliminate the firstpossible ATG (C instead of G) and two other changes to form the NdeIsite (CA instead of AG).

FIG. 28. Construction of pS0DβMA

The entire coding region of human CuZn SOD on a NdeI-BamHI fragmentisolated from pSODα13, was inserted between the unique NdeI and BglIIsites of pβUN as depicted in FIG. 28. The resulting vector, pSODβMA, isan intermediate plasmid used in the construction of the expressionplasmids pS0D-βMAX₁₂ and pSOD-βMAX₁₀.

FIG. 29 Construction of pSOD-βMAX₁₂

The plasmid pSOD-βMAX₁₂ was constructed from pSOD-βMA by replacement ofpart of the ribosomal binding site with synthetic DNA. This causes asingle base change in the sequence of the ribosomal binding site. Theplasmid directs exclusive high level expression of a non-acetylated SODanalog which has an amino acid sequence identical to that of naturalSOD. The analog is also non-glycosylated. pSODβMAX₁₂ has been depositedwith the American Type Culture Collection and assigned ATCC AccessionNo. 67177.

FIG. 30 Construction of pSOD-βBMAX₁₀

The plasmid pSOD-βMAX₁₀ was constructed from pSOD-βMA by replacement ofpart of the ribosomal binding site with synthetic DNA. This causes asingle base change in the sequence of the riboseeal binding site. Theplasmid directs exclusive high level expression of a non-acetylated SODanalog which has an amino acid sequence identical to that of naturalSOD. The analog is also non-glycosylated.

FIG. 31 Modification of hSOD cDNA at 5' End and Construction ofpSODNH-10

Plasmid pS61-10 was digested with PstI and the small PstI-PstI 620 bpfragment containing hSOD DNA was isolated. The purified fragment wasseparated into two equal aliquots.

The first aliquot was treated with DNA polymerase (Klenow fragment) inorder to obtain a blunt-ended fragment. The DNA fragment was ligated toHindIII phosphorylated linkers having the sequence: ##STR10## andtreated with HindIII. The fragment was then ligated to pBR322 DNA whichhad been digested with HindIII and treated with bacterial alkalinephosphatase. The DNA ligation mixture was used to transform E colistrain 1061 and Amp^(R) clones were selected. These clones were screenedby in situ filter hybridization to a nicked translated radioactive probecontaining the hS0D DNA sequences. One of the positive clones, pS0DH3,was analyzed by restriction mapping.

The second aliquot of the 620 bp PstI-PstI fragment was digested withFokI and the 229 bp FokI-FokI fragment was isolated and ligated to asynthetic phosphorylated DNA linker having the sequence: ##STR11##

The DNA was then digested with NdeI and StuI. The newly formed 127 bpfragment with the synthetic linkers was ligated with T4 DNA ligase tothe large DNA fragment of plasmid pS0DH3 which was obtained by digestionwith NdeI, StuI and SalI. The NdeI-NdeI fragment of about 2560 bp wasisolated. The DNA ligation mixture was used to transform E. coli strain1061 and Amp^(R) transformants were selected. The clones were screenedfor the right plasmid construction by NdeI and HindIII double digestion.One of these clones (pSOD NH-10) was sequenced and used for theexpression of the human CuZn hS0D.

DETAILED DESCRIPTION OF THE INVENTION

A plasmid has been developed which enables the achievement of enhancedlevels of gene expression and polypeptide roduction. The plasmid is adouble-stranded DNA molecule. Upon introduction into a suitablebacterial host cell containing the thermolabile repressor C_(I) theplasmid renders the host cell capable, upon increasing the temperatureof the host cell to a temperature at which the repressor is inactivated,of effecting expression of a desired gene inserted into the plasmid andproduction of a polypeptide encoded by the gene.

The plasmid includes in 5' to 3' order the following:

a DNA sequence which contains the promoter and operator P_(L) O_(L) fromlambda bacteriophage;

the N utilization site for binding antiterminator N protein;

a first restriction enzyme site permitting replacement of the DNAsequence containing the ribosomal binding site which follows thereafter;

a DNA sequence which contains a ribosomal binding site for rendering themRNA of the desired gene capable of binding to ribosomes within the hostcell;

an ATG initiation codon or a DNA sequence which is converted into an ATGinitiation codon upon insertion of the desired gene into the vector;

a second restriction enzyme. site for inserting the desired gene intothe plasmid in phase with the ATG initiation codon; and

a gene encoding the desired polypeptide.

The plasmid also includes a DNA sequence which contains an origin ofreplication from a bacterial plasmid capable of automomous replicationin the host cell and a DNA sequence which contains a gene associatedwith a selectable or identifiable phenotypic trait which is manifestedwhen the plasmid is present in the host cell. The distance between the3' end of the P_(L) O_(L) promoter and operator sequence and the 5' endof the N utilization site is less than about 80 base pairs and thedistance between the 3' end of the N utilization site and the 5' end ofthe ribosomal binding site is less than about 300 base pairs.

Another component of the plasmid is a first restriction enzyme sitepermitting replacement of the DNA sequence containing the ribosomalbinding site which follows thereafter. Numerous such sites may be used.Suitable sites include EcoRI.

Yet another component of the plasmid is a second restriction enzyme sitefor insertion of the desired gene into the plasmid in phase with the ATGinitiation codon. Numerous such sites may be used. Suitable sitesinclude NdeI, ClaI, HindIII, SmaI, BglII, XbaI, SacI and AluI.

Generally it is desirable that the second restriction enzyme site alsofunctions as the second restriction site necessary to permit replacementof the DNA sequence containing the ribosomal binding site. If the secondrestriction site is not also used for this purpose then the vector ofthis invention must also include a third restriction enzyme site afterthe ribosomal binding site but prior to the second restriction site.

Preferably, the plasmid contains two unique restriction enzyme sites.The first site permits replacement of the DNA sequence containing theribosomal binding site. The second site permits insertion of the desiredgene into the plasmid in phase with the ATG initiation codon. The term"unique restriction enzyme" site as employed herein means a restrictionenzyme site which occurs only once in the plasmid. In a presentlypreferred embodiment, EcoRI is the first restriction enzyme site andNdeI is the second restriction enzyme site.

The preferred host for use with the plasmid is Escherichia coli. Thepresently preferred strains are A1637, A1645, A2602, A2097 and A1563.A1637 was obtained from C600 by inserting transposon containingtetracycline resistance gene within the galactose operon as well as thelambda system for expression which is close to galactose operon. c600 isavailable from the American Type Culture Collection, as ATCC AccessionNo. 23724.

A1645 was obtained from A1637 by selection for Gal⁺ (ability to fermentgalactose). as well as loss of tetracycline resistance. It stillcontains the lambda expression system but part of the transposon hasbeen removed by selection. Its phenotype is C600 r⁻ m⁺ gal⁺ thr⁻ leu⁻lacZ⁻ bl (λcI857 ΔH1 ΔBamH1 N⁺).

A1645 is presently a more preferred strain for expression of superoxidedismutase or an analog thereof. It has been deposited with the AmericanType Culture Collection in Rockville, Md., U.S.A. containing variousplasmids as described more fully hereinafter. All deposits were madepursuant to the Budapest Treaty on the International Recognition of theDeposit of Microorganisms except that pBR322 and pBRM are fullyavailable from the American Type Culture Collection as ATCC AccessionNos. 37017 and 37283, respectively, and D4 was deposited under ATCCAccession No. 31826 in connection with the filing of a U.S. patentapplication.

A2602 and A1563 are derived from SA500. Their phenotypes are SA500 his⁻ile⁻ gal⁺ Δ 8 (λcI857 ΔH1Δ Bam N⁺) and SA500 his⁻ ile⁻ gal⁺Δ8 lacZ⁻ A21αcI857 int2 xisl nutL₃ ΔHl), respectively. A2097 is derived from A1645.Its phenotype is A1645 lacΔX A21 proC: :Tn10.

Prototrophic strains of Escherichia coli which enable high levelpolypeptide expression even when grown in a minimal media may also beused as hosts for the vectors of this invention. Preferred prototrophicstrains include A4200 and A4255. Strain A4255 containing the plasmidp9200 has been deposited with the ATCC under Accession No. 53215. Evenmore preferred are biotin independent prototrophic strains such as A4346containing the plasmid pGH44 which has been deposited with the ATCCunder Accession No. 53218.

Lytic strains of Escherichia coli may also be used as hosts for thevectors of this invention. Suitable lytic strains include those whichproduce, at the temperature at which the polypeptide is produced but ata rate slower than that at which the polypeptide is produced, asubstance e.g., an enzyme like endolysin which will cause the cell tolyse. This permits the cell to produce relatively large amounts of thedesired polypeptide before the amount of the lysing substance producedreaches the level which causes cell lysis. Examples of suitable lyticstrains include those containing the PlcI^(ts) plasmid such as strainA4048 containing pGH44 which has been deposited with the ATCC underAccession No. 53217.

Preferably, the plasmid is a covalently closed circular double-strandedmolecule. However, it is not essential that the plasmid be covalentlyclosed.

The plasmid achieves its enhanced expression levels after the host cellis heated to a temperature at which the C_(I) repressor protein isdestroyed. A temperature above about 38° C. is effective for thispurpose and since it is desired that unnecessary heat damage to the hostcells be avoided to as great an extent as possible, it is generallydesirable that the temperature not exceed 42° C. by more than a fewdegrees.

One important component of the vector is the ribosomal binding site.Suitable sites are C_(II) from lambda bacteriophage having the sequence:##STR12## a mutant of C_(II) from lambda bacteriophage having thesequence: ##STR13## the major head protein gene of bacteriophage lambdahaving the sequence: ##STR14## the natural β-1actamase ribosomal bindingsite derived from pBR322; a synthetic oligonucleotide having thesequence: ##STR15## a synthetic oligonucleotide having the sequence:##STR16## a natural ribosomal binding site derived from Bacillusthurengensis.

The plasmid also includes an origin of replication from a bacterialplasmid capable of autonomous replication in the host cell. Suitablesuch origins of replication may be obtained from a number of sources,e.g., from pBR322 or pR1.

A DNA sequence which contains a gene associated with a selectable oridentifiable phenotypic trait which is manifested when the plasmid ispresent in the host cell is also a component of the plasmid. Suitablegenes include those associated with temperature sensitivity or drugresistance, e.g., resistance to ampicillin, chloroamphenicol ortetracycline.

Relative to plasmids described previously, the plasmids of thisinvention may be used to obtain enhanced expression of a wide variety ofgenes encoding desirable polypeptide products. Suitable genes includethose encoding growth hormones, e.g. , bovine, porcine, chicken or humangrowth hormones; superoxide dismutase; apolipoprotein E or analogs ofany of the preceding. By analog is meant a polypeptide having the sameactivity as the naturally occurring polypeptide but having one or moredifferent amino acids added or deleted, or both, at the N-terminus ofthe polypeptide.

The plasmid may be formed by methods well known to those of ordinaryskill in the art to which the invention relates. Such methods aredescribed in greater detail in various publications identified herein,the contents of which are hereby incorporated by reference into thepresent disclosure in order to provide complete information concerningthe state of the art.

One presently preferred plasmid is pJH200 which has the restriction mapshown in FIG. 2. This plasmid was introduced into Escherichia coli usinga strain A1645 conventional transformation method. The resulting hostvector system has been deposited under ATCC Accession No. 39783. A geneencoding a desired polypeptide, e.g. bovine growth hormone, may beinserted into pJH200.

A second preferred plasmid, pR0211, was constructed from a partial NdeIdigest of pJH200. pR0211 has the re striction map shown in FIG. 2.Bovine growth hormone cDNA has been inserted into pR0211 by digestingthe vector with NdeI and HindIII, isolating the large fragment andligating to it bGH cDNA obtained from pAL500 (ATCC Accession No. 39782).The resulting plasmid is designated pR012. Its restriction map is alsoshown in FIG. 2.

Plasmid pR012 has been partially digested with PvuII followed by NdeI. Asynthetic DNA fragment coding for the first 24 amino acids of theN-terminus of authentic bGH has been ligated to the digested pRO12. Theresulting plasmid, designated pSAL 5200-6, has the restriction map shownin FIG. 3.

The plasmids of this invention may also be engineered to produce humansuperoxide dismutase (SOD) analogs thereof or mixtures of SOD analogs. Afragment of pJH200 (ATCC Accession No. 39783) containing the λP_(L)promoter and C_(II) ribosomal binding site was isolated and theninserted into a plasmid pSOD NH-10 which contains the gene for human SODto form a plasmid designated pSOD NH-550 as shown in FIG. 13. A fragmentcontaining both the λP_(L) promoter and the SOD gene was isolated frompSOD NH-550 following digestion with AluI. After the addition of BamHIlinkers and subsequent restriction with BamHI, the fragment was insertedinto the unique BamHI site of pBRM. pBRM is a high copy number plasmidwhich has been deposited under ATCC Accession No. 37283. The resultingplasmid is designated pSODα2. It has the restriction map shown is FIG.13. This plasmid has been deposited in E. coli strain A2097 under ATCCAccession No. 39786.

Plasmid pSODα2 (ATCC Accession No. 39786) contains the C_(II) ribosomalbinding site. This ribosomal binding site has been replaced with afragment containing the β-lactamase promoter and Shine-Dalgarnoribosomal binding site isolated from an EcoRI-AluI digest of pBLA11.(Plasmid pBLA11 has the restriction map shown in FIG. 14 and has beendeposited in Escherichia coli strain A1645 under ATCC Accession No.39788.) The C_(II) ribosomal binding site is removed from plasmid pSODα2as shown in FIG. 14. pSODα2 is partially restricted with EcoRI, filledin with DNA poly-merase I (Klenow) and religated, so that the onlyremaining EcoRI site in the plasmid is located at the 5' end of theC_(II) RBS. The resulting plasmid, designated pSODα13 was digested withNdeI, filled in with DNA polymerase I (Klenow) and then digested withEcoRI. The large fragment was isolated and ligated to the fragmentcontaining the β-lactamase promoter and ribosomol binding site isolatedfrom pBLA11 to form plasmid pSOD β1.

pSODβ1 may be modified to include a tetracycline resistance genefragment (Tet^(R)) instead of an ampicillin resistence gene fragment(Amp^(R)). The Amp^(R) fragment was removed from pSOD 1 by digestionwith PstI followed by partial BamHI. The resulting plasmid was filled inwith DNA polymerase I (Klenow). The Tet^(R) gene fragment was separatelyisolated from an EcoRI-AvaI digest of pBR322, filled in and ligated tothe filled in plasmid. (Plasmid pBR322 is widely available, e.g. fromthe American Type Culture Collection as ATCC Accession No. 37017). Thethen resulting plasmid is designated pSODβ₁ T₁₁. It has the restrictionmap shown in FIG. 15 and has been deposited with the American TypeCulture Collection and assigned Accession No. 53468. The plasmid pSOD β₁T₁₁ produces a mixture of two hSOD analogs: (1) the non-acetylatedanalog of human SOD having an amino acid sequence identical to that ofnatural human CuZn superoxide dismutase (non-acetylated hCuZn SODanalog); and (2) the non-acetylated analog of human SOD having the aminoacid sequence identical to that of natural human CuZnSOD and having theamino acid sequence ser-met attached to the amino terminus(non-acetylated ser-met hCuZnSOD analog).

The composition of the mixture may range from about 90% to about 95% byweight of the non-acetylated hCuZnSOD analog and from about 5% to about10% by weight of the non-acetylated ser-met hCuZnSOD analog. A preferredcomposition is one wherein the non-acetylated hCuZnSOD analog is greaterthan about 93% by weight of the mixture and the non-acetylated ser-methCuZnSOD analog is less than about 7% by weight of the mixture. A morepreferred composition is one wherein the non-acetylated hCuZnSOD analogis approximately 95% by weight of the mixture and the non-acetylatedser-met hCuZnSOD analog is approximately 5% by weight of the mixture.

Other plasmids which may be used to produce human superoxide dismutaseor analogs thereof are pSODβ MAX₁₂ and pSODβMAX₁₀. The construction ofthese plasmids is shown in FIGS. 26-30. Each of these plasmidsexclusively produces the non-acetylated hSOD analog having an amino acidsequence identical to that of natural human CuZnSOD.

One further plasmid which may be used to produce human superoxidedismutase is designated pSOD β₁ -BA2. Its construction from pSODα13 isshown in FIG. 17.

The vector of this invention, e.g. pR0211 may also be engineered toproduce porcine or chicken growth hormones. Thus, as shown in FIG. 4,porcine growth hormone cDNA was isolated from an NdeI digest ofppGH-NdeI/RI. The resulting fragment containing the pGH gene was ligatedto an NdeI digest of pR0211. The resulting plasmid, designated p3008,has been deposited in E. coli strain A2097 under ATCC Accession No.39804.

In another embodiment of the invention two chicken growth hormonefragments were isolated from NdeI-BamII digest of pcGH-NdeI/RI as shownin FIG. 5. The two cGH fragments were ligated to a phosphorylatedsynthetic linker which Codes for the first 18 amino acids of theN-terminus of authentic cGH. The sequence of the linker was: ##STR17##

The resulting fragment was then ligated to a NdeI digest of pR0211 toform the plasmid designated p5002 which has the restriction map shown inFIG. 5.

The vectors of this invention may also be engineered to produce humanapolipoprotein E. The gene for human apoplipoprotein E (ApoE) may beisolated from plasmid pApoE-EX2 by NdeI digestion. pApoE-Ex2 has therestriction map shown in FIG. 18. It has been deposited in E. colistrain A1645 under ATCC Accession No. 39787.

The ApoE gene (cDNA) may be placed in various plasmids. Among thepreferred embodiments is plasmid pTV-188 which has the restriction mapshown in FIG. 18. pTV-188 was constructed by ligation of the ApoE geneisolated from pApoE-Ex2 to a StuI digest of plasmid pSODβ₁ T₁₁. pTV-188contains the Tet^(R) fragment, the λP_(L) promoter sequence, theβ-lactamase promoter and Shine-Dalgarno sequence. This plasmid expressesan ApoE fused protein.

Another preferred embodiment of a plasmid which contains the ApoE geneis pSAL 160-5 which has the restriction map shown in FIG. 23. pSAL 160-5was constructed from pTV 104(2) (ATCC No. 39384) and plasmid pTV-170,(see also FIG. 20). The ApoE gene was isolated from pTV-170 and insertedinto pTV 104(2) at the pstI site within the human growth hormone genesequence. The resulting plasmid pSAL 160-5 contains the Amp^(R) fragmentand the λ P_(L) promoter sequence.

10 Using the same approach other plasmids may be prepared by replacingthe gene encoding the desired polypeptide at the second restrictionenzyme site of the plasmid.

Various host vector systems involve E. coli A1637, Al645, A2606, A2097,A1563, A4200, A4255 and A4048 and the plasmid described herein may beused to produce different polypeptides such as bovine, porcine, chickenand human growth hormones, human superoxide dismutase and humanapoliprotein E. To do so, the host vector system is grown under suitableconditions permitting production of polypeptide which is then recovered.

Suitable conditions involve growth of the host vector system for anappropriate period of time at about 42° C. Desirably, the period ofgrowth at 42° C. is about 1 to 5 hours. Suitable media include caseinhydrolysate.

By means of the preceding method a number of bGH, pGH, cGH, ApoE and SODanalogs have been prepared.

ApoE analogs have been prepared which have an amino acid sequenceidentical to that of natural ApoE except for variations at theN-terminus. Examples include the following:

1) amino acid methionine added to N-terminus of natural humanapolipoprotein E;

2) natural human apolipoprotein E to the N-terminus of which is attachedthe 42 amino acid N-terminal sequence of human superoxide dismutase andthen methionine; and

3) natural human apolipoprotein from which the 11 N-terminal amino acidshave been deleted and replaced by the 45 amino acid N-terminal sequenceof mature human growth hormone followed by methionine.

A pGH analog has been prepared in which the amino acid methionine isadded to the N-terminus of natural porcine growth hormone.

A cGH analog has been prepared in which the amino acid methionine isadded to the N-terminus of natural chicken growth hormone.

SOD analogs have been prepared which have an amino acid sequenceidentical to that of natural SOD except for variations at theN-terminus. Examples include the following:

1) natural human SOD which is non-acetylated;

2) natural human SOD which is non-acetylated and non-glycosylated;

3) natural human SOD which is non-acetylated and has the ser-met aminoacid sequence attached to the amino terminus; and

4) natural human SOD which is non-acetylated and non-glycosylated andhas the ser-met amino acid sequence attached to the amino terminus.

These SOD analogs or a mixture of same may be used to catalyze thedismutation or univalent reduction of the superoxide anion in thepresence of proton to form hydrogen peroxide as shown in the followingequation: ##STR18## Veterinary compositions may be prepared whichcontain effective amounts of one or more bGH, cGH or pGH analogs and asuitable carrier. Such carriers are well known to those of ordinaryskill in the art. The analogs may be administered directly or in theform of a composition to a cow in order to increase milk or meatproduction, to a chicken in order to increase meat production or to apig in order to increase meat production.

Pharmaceutical compositions may be prepared which contain effectiveamounts of one or more ApoE analogs and a suitable carrier. Suchcarriers are well known to those skilled in the art. The analogs may beadministered directly or in the form of a composition to a humansubject, e.g., to treat deficiencies in ApoE production by the subject,or to treat atherioscelerosis.

Veterinary and pharmaceutical compositions may also be prepared whichcontain effective amounts of SOD or one or more SOD analogs and asuitable carrier. Such carriers are well-known to those skilled in theart. The SOD or analog may be administered directly or in the form of acomposition to the animal or human subject, e.g., to treat a subjectafflicted by inflammations or to reduce injury to the subject byoxygen-free radicals on reperfusion following global ischemia or organtransplantation e.g., kidney transplantation. The SOD or analog may alsobe added directly or in the form of a composition to the perfusionmedium of an isolated organ, e.g., to reduce injury to an isolated organby oxygen-free radicals on perfusion after excision, thus prolonging thesurvival period of the organ, e.g. cornea. Additionally, the SOD oranalog may be used to reduce spinal injury and for bronchial pulmonarydysplasia.

Examples

The examples which follow are set forth to aid in understanding theinvention but are not intended to, and should not be construed to, limitits scope in any way. The examples do not include detailed descriptionsfor conventional methods employed in the construction of plasmids, theinsertion of genes encoding polypeptides of interest into such plasmidsor the introduction of the resulting plasmids into bacterial hosts. Suchmethods are well known to those of ordinary skill in the art and aredescribed in numerous publications including by way of example thefollowing:

T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning; ALaboratory Manual, Cold Spring Harbor Laboratory, New York (1982).

Methods in Enzymology, vol. 65, "Nucleic Acids (Part 1), " edited byLawrence Grossman and Kivie Moldave, Academic Press New York (1980).

Methods in Enzymology, vol. 68, "Recombinant DNA, edited by Ray Wu,Academic Press, New York (1981).

Methods in Enzymology, vol. 100, "Recombinant DNA (Part B)," edited byRay Wu, Lawrence Grossman and Kivie Moldave, Academic Press, New York(1983).

Methods in Enzymology, vol. 101, "Recombinant DNA (Part C)," edited byRay Wu, Lawrence Grossman and Kivie Moldave, Academic Press, New York(1983).

Principles of Gene Manipulation, An Introduction to Genetic Engineering,2nd Edition, edited by R. W. Old and S. B. Primrose, University ofCalifornia Press (1981).

H. V. Bernard, et al., Gene (1979) 5, 59.

A. B. Oppenheim, et al., J. Mol. Biol. (1982) 158, 327.

E. Remaut, et al., Gene (1981) 15, 81.

EXAMPLE 1 Expression vectors

As used herein the term "expression vector" refers to a group ofplasmids useful for expressing desired genes in bacteria, particularlyin E. coli. The desired gene may be inserted into the expression vectoror alternatively, the promoters on the expression vector may be excisedand placed in front of the desired gene.

pJH200

pJH200, shown in FIG. 2, is composed of a DNA inserted into themulticopy plasmid pBR322. The salient features of the λDNA are that itcontains the XλP_(L) promoter, the leftward N utilization site(nut_(L)), an EcoRI restriction site, the t_(Ri) termination site,followed by the C_(II) ribosomal binding site and an ATG initiationcodon which is part of the NdeI restriction site. One hundred andsixteen (116) base pairs downstream Of the NdeI restriction site arefour unique restriction sites as shown in FIG. 2. The restriction sitesenable facile insertion of the desired gene. The C_(II) ribosomalbinding site differs from the natural ribosoma binding site by a singlepoint mutation.

pJH200 was constructed from pOG11 (A. Oppenheim, etal., J. Mol. Biol.(1982) 158; 327) and contains the XλP_(L) promoter and the C_(II)ribosomal binding site found in pOGll. However, 346 bp of λDNA locatedbetween the λP_(L) promoter and the C_(II) ribosomal binding site havebeen deleted, and an EcoRI restriction site has been introduced at thejunction between these two elements. Also, a multi-restriction sitelinker was introduced "downstream" of the ribosome binding site. pJH200has been deposited with the American Type Culture Collection under ATCCNo. 39783.

PRO211, shown in FIG. 2 and described in detail in the Description ofFigures, was derived from pJH200 by eliminating one of the two NdeIrestriction sites.

pJH200, pRO211 and derivatives thereof containing eucaryotic genes maybe maintained in suitable E. coli hosts. The most important feature of asuitable host is that it provide the thermosensitive repressor cI857 andthe antitermination N protein. (M. E. Gottesman, et al., J. Mol. Biol.(1980) 140; 57-75).

pRO211 has numerous advantages over previously described expressionvectors including:

1. extremely high levels of expression

The vector is capable of directing expression of foreign proteins in E.coli. at levels as high as 35% of the total cellular protein.

2. replaceable ribosomal binding site

pRO211 contains a unique EcoRI site which is located "upstream" of theribosomal binding site, and an NdeI site located "downstream" of theribosomal binding site. Thus, the ribosomal binding site is bounded bytwo unique restriction sites. This enables facile excision of thepresent ribosomal binding site (the λC_(II) ribosomal binding site) andsubstitution of virtually any other natural or synthetic ribosomalbinding site without altering other features of the plasmid. Thisgreatly facilitates optimal expression of desired polypeptides.

3. thermoinducible regulation of expression

The λP_(L) promoter is inactive when the C_(I) repressor is bound to it.The cI857 repressor is thermosensitive, that is, it binds to thepromoter at 30° C. but is inactivated at 42° C. Thus, by increasing thetemperature of fermentation to 42° C. the host bacteria are induced toproduce the desired protein.

The advantages of such a system include the following:

(a) A foreign protein which is toxic to Escherichia coli can be producedlate in the fermentation process thus avoiding early cell death,

(b) Overproduction of a protein may stabilize the protein and preventproteolytic degradation. (Cheng, Y. E., et al., Gene (1981) 14, 121).Thus, "instantaneous" overproduction using a tightly regulated promotersuch as λP_(L) may be preferable to continuous low level production.

4. simplified induction protocol

Protein production by the plasmids described in this patent applicationand in copending, coassigned U.S. patent application Ser. No. 514,188 isregulated by the thermosensitive cI857 repressor.

The induction protocol required by the plasmids described in thecopending, coassigned application involved induction at 42° C. followedby growth at 38° C. In contrast, the optimal induction of proteinsynthesis when using the vectors pJH200, pRO211 or their plasmidderivatives involved induction at 42° C. followed by growth at the sametemperature, i.e. 42° C. This eliminates the need to cool the fermentor.

5. copy number

The λP_(L) promoter in pJH200 and pRO211 is found on a plasmid with acopy number higher than the λ transducing phage vectors which arepresent in E. coli. This increases expression levels.

6. ribosome binding site and initiation codon

This expression vector contains a strong procaryotic ribosomal bindingsite (RBS) as well as a translation initiation codon (ATG). Thus, anyeucaryotic gene may be cloned without adding the initiation codon.Furthermore, the efficient RBS increases levels of expression. Theribosome binding site is the λC_(II) ribosomal binding site. Thesequence of the ribosomal binding site is: ##STR19##

One base pair is different from the ribosomal binding site found in thewild type λ.

7. convenient restriction site

The expression vector has a unique NdeI restriction site which containswithin the site the ATG initiation codon. This permits properpositioning of the desired gene. The unique NdeI site is foundimmediately after the ribosomal binding site.

8. convenient restriction sites for gene insertion

Located 116 base pairs downstream of the NdeI restriction site are 4other unique restriction sites in the following order: BglII, SmaI,HindIII and ClaI. The multiplicity of unique restriction sites enablesfacile insertion of desired genes.

9. nut site

N protein, which is provided by the host, binds the Nut site on theexpression vector and thereby prevent termination of transcription atthe t_(RI) site or premature transcription termination within the clonedgene.

Strains

Suitable hosts for the described vectors and plasmids are strains of E.coli suitable for transformation, including A1637, A2602, A1563, A1645(C600 r⁻ m⁺ gal⁺ thr⁻ leu⁻ lac⁻ bl (λcI857 ΔHl ΔBamHI N⁺)) and A2097(A1645 lac ΔXA21 proC::Tn 10).

EXAMPLE 2 Animal Growth Hormones

I. pRO12

The construction of pRO12 is shown in FIG. 2 and described in theDescription of the Figures. bGH cDNA from pAL500 whose construction isshown in FIG. 1, was manipulated prior to insertion into pRO211 toprovide the correct reading frame and an NdeI restriction site.

pRO12 was introduced into EscheriChia coli strain A1645 bytransformation using methods known to those of ordinary skill in theart. This strain produces upon growth and induction an analog of bovinegrowth hormone (bGH) having the amino acid sequence met-asp-gln added tothe N-terminus of the phenylalanine form of natural bGH. The amount ofbGH analog produced by pRO12 was about 30-36% of the total proteinproduced by the bacteria as calculated by scanning Coomasie blue-stainedSDS polyacrylamide gels (Table I).

II. pSAL 5200-6

The construction of pSAL 5200-6 is shown in FIG. 3 and described in theDescription of the Figures. The DNA sequence coding for met-phe bGH wasobtained by restricting pRO12 with PvuII and NdeI and inserting asynthetic DNA fragment formed from two single-stranded syntheticoligonucleotides having 10 base pair overlapping segments.

pSAL 5200-6 was introduced into Escherichia coli strain A1645 bytransformation using known methods. This strain produces upon growth andinduction an analog of bGH having a methionine added to the aminoterminus of phe bGH. The amount of the met-phe bGH analog produced bypSAL 5200-6 was about 18-20% of the total protein produced by thebacteria as calculated from scanning Coomasie-stained SDS polyacrylamidegels. The methods used to grow the strain, recover the bGH produced andpurify the bGH are the same as those described hereinafter in Example 5for bGH production from pRO12.

III. p3008

The construction of p3008 is shown in FIG. 4 and described in theDescription of the Figures. p3008 has been deposited with the AmericanType Culture Collection under ATCC No. 39804. The DNA sequence codingfor met-phe pGH (porcine growth hormone) was obtained by inserting pGHcDNA into pRO211.

p3008 was introduced into Escherichia coli strain A1645 bytransformation using methods known to those of ordinary skill in theart. This strain produces upon growth and induction pGH having amethionine added to the amino terminus of phe pGH. The amount of themet-phe pGH analog produced by p3008 was about 18-20% of the totalprotein produced by the bacteria as calculated from scanningCoomasie-stained SDS polyacrylamide gels. The methods used to grow thestrain, recover the pGH produced and purify the pGH are the same asthose described hereinafter in Example 5 for bGH production from pRO12.

IV. p5002

The construction of p5002 is shown in FIG. 5 and described in theDescription of the Figures. The DNA sequence coding for met-phe cGH(chicken growth hormone) was obtained by inserting cGH cDNA into pRO211and completing the 5' end of the gene with synthetic oligonucleotideslinkers.

p5002 was introduced into Escherichia coli strain A1645 bytransformation using known methods. This strain produces upon growth andinduction cGH having a methionine added to the amino terminus of phecGH. The amount of the met-phe cGH analog produced by p5002 was about18-20% of the total protein produced by the bacteria as calculated fromscanning the Coomasie-stained SDS polyacrylamide gels. The methods usedto grow the strain, recover cGH produced and purify the cGH are the sameas those described hereinafter in Example 5 for bGH production frompRO12.

                  TABLE I.sup.1                                                   ______________________________________                                                             %                                                        Plasmid    Host      bGH.sup.2                                                                             Remarks                                          ______________________________________                                        pRec 2/3   A1637     23      Amp.sup.R                                        pRO11      A1637     28      Amp.sup.R                                        pRO12      A1645     30-36   Amp.sup.R                                        pHG44      A2097     37-42   Amp.sup.R,T.sub.1 T.sub.2                        pHG50      A1645     37-42   Amp.sup.R,T.sub.1 T.sub.2 ;cI.sup.434            pSAL-130/5 A1645     39-44   Amp.sup.R ;CHCN;T.sub.1 T.sub.2                  pSAL-170/10                                                                              A1645     40-46   Tet.sup.R ;CHCN;T.sub.1 T.sub.2                  ______________________________________                                         .sup.1 The table summarizes the bGH expression levels of various plasmids     derived from pRO211. The plasmids pRec 2/3 and pRO11 are described in         copending, coassigned U.S. Pat. Appl. Ser. No. 514,188, filed July 15,        1983.                                                                         .sup.2 Amount of bGH produced as percentage of total bacterial protein.  

1. The table summarizes the bGH expression levels of various plasmidsderived from pRO211. The plasmids pRec 2/3 and pRO11 are described incopending, coassigned U.S. patent application Ser. No. 514,188, filedJul. 15, 1983.

Amount of bGH produced as percentage of total bacterial protein.

EXAMPLE 3 Human Cu-Zn Superoxide Dismutase (SOD)

The starting point for Cu-Zn SOD cDNA modifications is the plasmidpS61-10 described in Lieman-Hurwitz, J., et al., PNAS (1982), 79: 2808.The SOD cDNA is also described in copending U.S. patent application Ser.No. 489,786, filed Apr. 29, 1983. The SOD cDNA was modified to introducean NdeI restriction site at the 5' end of the gene and a HindIIIrestriction site at the 3' end of the gene. The resulting plasmid,pSODNH-10, contains SOD cDNA bounded by unique restriction sites.

Modification of hSOD cDNA at 5'-End-and Construction of pSODNH-10

The modification of hSOD cDNA at the 5'-end and construction ofpSODNH-10 is shown in FIG. 31 and described in the Description of theFigures.

Plasmid pS61-10 was digested with PstI and the small PstIPst. PstI 620bp fragment containing hSOD DNA was isolated by electrophoresis on 1%agarose gel. The purified fragment was dissolved in 50 ul of TE bufferand separated into two equal aliquots. The first aliquot was treatedwith DNA polymerase (Klenow fragment) in the presence of all 4 dNTP's inorder to obtain a blunt-ended fragment. The Klenow reaction was carriedout at 16° C. for 30 minutes and the reaction was terminated byphenol-chloroform extraction and ethanol precipitation. The DNA fragmentwas dissolved in ligation buffer and was ligated to HindIIIphosphorylated linkers with the sequence: ##STR20##

The reaction was terminated by heating at 70° C. for 5 minutes, andtreated with HindIII (100 units). The fragment was then ligated topBR322 DNA which was digested with HindIII (100 units). The fragment wasthen ligated to pBR322 DNA which was digested with HindIII and treatedwith bacterial alkaline phosphatase. The DNA ligation mixture was usedto transform E. coli strain 1061 and Amp^(R) clones were selected. Theseclones were screened by in situ filter hybridization to a nickedtranslated radioactive probe containing the hSOD DNA sequences. One ofthe positive clones, pSODH3, was analyzed by restriction mapping.

The second aliquot of the 620 bp PstI-PstI fragment was digested withFokI and the 229 bp FokI-FokI fragment was purified by electrophoresison a 1% agarose gel redissolved in ligation buffer and ligated to asynthetic phosphorylated DNA linker with the sequence: ##STR21##

The reaction was stopped by heating at 70° C. for 5 minutes and then theDNA was digested with NdeI and StuI. The newly formed 127 bp fragmentwith the synthetic linkers was ligated with T4 DNA ligase to the largeDNA fragment of plasmid pSODH3 which was obtained by digestion withNdeI, StuI and SalI: NdeI-StuI fragment of about 2560 bp was isolated.The DNA ligation mixture was used to transform E. coli strain 1061 andAmp^(R) transformants were selected. The clones were screened for theright plasmid construction by NdeI and HindIII double digestion. One ofthese clones (pSOD NH-10) was sequenced and has been used for theexpression of the human CuZn hSOD.

I. pSODα2

The construction of pSODα2 is shown in FIG. 13 and described in theDescription of the FIGS. pSODα2 has been deposited with the AmericanType Culture Collection under ATCC No. 39786. To construct pSODα2, theλP_(L) promoter, the Nut_(L) and the C_(II) ribosomal binding site wereexcised from the expression vector pJH200 and placed in front of the SODgene of plasmid pSOD NH-10. Then, the fragment containing both thepromoter, the MS and the SOD gens was inserted into the vector pBRM(Hartman, J. R., et al., PNAS 79:233-237 (1982). pBRM has been depositedwith the American Type Culture Collection under ATCC No. 37283.

pSODα2 was introduced into Escherichia coil-strain A2097 bytransformation using known methods. The clones obtained produce upongrowth and induction an SOD analog protein. The amount of SOD analogproduced by pSODα2 was about 0.1-0.3% of the total protein produced bythe bacteria as calculated from scanning of Coomasie-stained SDSpolyacrylamide gels (Table II). The SOD analog produced is probablyidentical to that produced by pSOD81 described in the followingparagraph.

II. pSODβ1

The construction of pSODβ1 is shown in FIG. 14 and described in theDescription of the Figures. To construct pSODβ1, the C_(II) RBS ofpSODα2 was replaced with the β-lactamase promoter and RBS derived frompBLA11. pBLA11 has been deposited with the American Type CultureCollection under ATCC No. 39788. pBLA11 contains the promoter andribosomal binding site of the 3-lactamase gene found in pBR322 betweencoordinates 4157 and 4353. An EcoRI linker was added upstream of thepromoter and a multi-restriction site linker was added immediately afterthe initiation codon ATG. Thus, the sequence of the coding strandbeginning with the initiation codon is ATGAGCTCTAGAATTC.

pSODβ1 was introduced into Escherichia coli strain A1645 bytransformation using known methods. The clones obtained produce upongrowth and induction an SOD analog. The human Cu-Zn SOD analog produceddiffers from natural human Cu-Zn SOD in that the amino terminus alanineis not acetylated, as demonstrated by amino acid sequencingstoichiometry while the natural human SOD is acetylated at the aminoterminus alanine (Hartz, J. W. and Deutsch, H. F., J. Biol. Chem. (1972)234:7043-7050; Jabusch, J. R., et al., Biochemistry (1980) 19:2310-2316;Barra, et al., FEBS Letters (1980) 120:53 and Oberley, L. W., SuperoxideDismutase, Vol. I, (1982), CRC Press, Florida, pp. 32-33.). Furthermore,the natural human SOD is glycosylated (Huber, W., U.S. Pat. No.3,579,495, issued May 18, 1971) while bacterial-produced human SOD isalmost certainly not glycosylated, because Escherichia coli does notglycosylate proteins which it produces. The amino acid sequence of thebacterial-produced SOD analog is identical to that of mature human SODand does not contain a methionine residue at its N-terminus.

The amount of SOD produced by pSODS1 was about 3-8% of the total proteinproduced by the bacteria as calculated from scanning of Coomasie-stainedSDS polyacrylamide gels (Table II). The methods used to grow the strain,recover the SOD produced and purify the SOD are the same as thosedescribed hereinafter in Example 7 for pSOD β₁ T₁₁.

III. pSOD.sub.β1 T₁₁

The construction of pSOD.sub.β1 T₁₁ is shown in FIG. 15 and described inthe Description of the Figures. The gene coding for ampicillinresistance of pSODα1 was replaced with the gene coding for tetracyclineresistance derived from pBR322. pSOD.sub.β1 T₁₁ has been deposited withthe American Type Culture Collection and assigned ATCC Accession No.67177. The amount of SOD analog produced by pSODβ₁ T₁₁ was about 8-13%of the total protein produced by the bacteria as calculated fromscanning of Coomasie-stained SDS polyacrylamide gels (Table II). The SODproduced by pSODβ₁ T₁₁ is a mixture of SOD analogs. More than about 93%by weight of the SOD produced by pSODβ₁ T₁₁ is the non-acetylated formof natural human superoxide dismutase having an amino acid identical tothat of natural human CuZn superoxide dismutase and less than 7% byweight is the non-acetylated form of natural human superoxide dismutasehaving an amino acid sequence identical to that of natural human CuZnsuperoxide dismutase and having the amino acid sequence ser-met at theamino terminus. As used throughout this specification, the term SODanalog produced by pSODβ₁ T₁₁ means a mixture of these SOD analogs.

IV. pSODβ₁ -BA2

The construction of pSODβ₁ -BA2 is shown in FIG. 17 and described in theDescription of the Figures. The C_(II) ribosomal binding site of pSODα13was replaced by a synthetic DNA fragment with the sequence: ##STR22##which is similar to the sequence of the natural 3-lactamase RBS. pSODβ₁-BA2 was introduced into Escherichia coli strain A1645 by transformationusing methods known to those of ordinary skill in the art. The clonesobtained produce upon growth and induction an analog of human SOD. Theamount of SOD produced by pSODβ₁ -BA2 was about 2-4% of the totalprotein produced by the bacteris as calculated from scanning ofCoomasie-stained SDS polyacrylamide gel (Table II). The SOD analogproduced is identical to that produced by pSODβ1.

                  TABLE II                                                        ______________________________________                                        Plasmid   RBS       Host    %SOD.sup.3                                                                            Remarks                                   ______________________________________                                        pSODα2                                                                            C.sub.II  A2097   0.1-0.3 Amp.sup.R                                 pSODβ.sub.1                                                                        BLA.sup.1 A1645   3-8     Amp.sup.R                                 pSODβ.sub.1 T.sub.11                                                               BLA.sup.1 A1645    8-13   Tet.sup.R                                 pSODβ.sub.1 TT-1                                                                   BLA.sup.1 A1645   10-15   Tet.sup.R ;T.sub.1 T.sub.2                pSODβ.sub.1 -BA2                                                                   BLA.sup.2 A1645   2-4     Amp.sup.R                                 ______________________________________                                         .sup.1 Promoter and ribomosal binding site of lactamase gene.                 .sup.2 Synthetic ribosomal binding site corresponding to that of the          lactamase gene.                                                               .sup.3 Amount of SOD analog produced expressed as percentage of total         bacterial protein.                                                       

ABBREVIATIONS

Amp^(R) =Ampicillin resistance

Tet^(R) =Tetracycline resistance

T₁ T₂ =Transcription termination sequences

EXAMPLE 4 Human Apolipoprotein E (ApoE3)

The starting point for ApoE3 cDNA modifications was the plasmid pNB178provided by Dr. John Taylor of the Gladstone Foundation, San Francisco,Calif. This plasmid contains a full length cDNA copy of the human ApoE3gene. The cDNA in pNB178 was modified to remove noncoding DNA at the 5'end of the gene and to add NdeI restriction sites at both ends of thegene. This ApoE3 cDNA fragment was inserted into the vector pND5(described in copending, coassigned U.S. patent application Ser. No.514,188, filed-Jul. 15, 1983). The resulting plasmid, pApoE-Ex2, shownin FIG. 18, has been deposited with the American Type Culture Collectionunder ATCC No. 39787.

I. pTV-188

The construction of pTV-188 is shown in FIG. 18 and described in theDescription of the Figures. The plasmid pTV188 was obtained by insertionof the NdeI-NdeI filled-in, ApoE3 fragment into the unique blunt end StuI site of pSODβ₁ T₁₁ (shown in FIG. 14 and described in the Descriptionof the Figures.)

pTV-188 was introduced into Escherichia coil strain A1645 bytransformation using methods Known to those of ordinary skill in theart. The clones obtained produce upon growth and induction an analog ofhuman ApoE3 having 42 amino acids of the N-terminal sequence of humansuperoxide dismutase attached to the N-terminus of authentic human ApoE3followed by methionine at the N-terminus of the analog. The ApoE3 analogproduced was about 10% of the :oral protein produced by the bacteria ascalculated by scanning Coomasie-stained SDS polyacrylamide gels. Themethod used to grow the strain is the same as that described in Example5 for bGH production from pRO12 except that 12.5 mg/liter tetracyclineis used instead of ampicillin.

II. pSAL 160-5

The construction of pSAL 160-5 is shown in FIG. 23 and described in theDescription of the Figures. The plasmid pSAL 160-5 was obtained byinsertion of the AvaI-AvaI ApoE3 gene fragment from pTV-170 (see FIG.20).

pSAL 160-5 was introduced into Escherichia coli strain A1645 bytransformation using methods known to those of ordinary skill in theart. The clones obtained produce upon growth and induction and analog ofApoE3 which contains at its amino terminus methionine and then 45 aminoacids from the N-terminus of human growth normone fused to ApoE3 fromwhich the 11 N-terminal amino acids have been deleted. The amount ofApoE3 analog produced by pSAL 160-5 was about 5% of the total proteinproduced by the bacteria as calculated by scanning Coomasie-stained SDSpoiyacrylamids gels. The method used to grow the strain is the same asthat described in Example 5 for bGH production from pRO12.

EXAMPLE 5 Growth of pRO12 I. Stock Cultures

Stock cultures of pRO12 were grown on casein medium (see Inoculum), thendiluted two-fold with freezing medium and stored at -80° C. Freezingmedium contains per 500 ml:

    ______________________________________                                        K.sub.2 HPO.sub.4     6.3    gr                                               KH.sub.2 PO.sub.4     1.8    gr.                                              Na citrate            0.45   gr                                               MgSO.sub.4.7H.sub.2 O 0.09   gr                                               (NH.sub.4).sub.2 SO.sub.4                                                                           0.9    gr                                               Glycerol              44.0   gr                                               ______________________________________                                    

II. Inoculum

The inoculum was propagated in 20 g/l casein hydrolysate, 10 g/l yeastextract and 2 g/l NaCI. Sterile medium in a shake flask was inoculatedfrom stock culture and incubated 15 hours on a shaker at 30° C. andapproximately 200 r.p.m. As needed subsequent stages in inoculumpropagation were carried out in stirred aerated fermenters. Sterilemedium was inoculated with 2-10% inoculum and incubated 15 hours at 30°C., pH 7±0.5 with agitation and aeration to maintain a dissolved oxygenlevel above 20% air saturation.

III. Production

The production medium contains:

    ______________________________________                                        Casein hydrolystae     20     g/l                                             Yeast extract          10     g/l                                             K.sub.2 HPO.sub.4      2.5    g/l                                             MgSO.sub.4.7H.sub.2 O  1      g/l                                             NaCl                   5      g/l                                             Biotin                 0.1    mg/l                                            Thiamine               1      mg/l                                            Trace elements solution                                                                              3      ml/l                                            ______________________________________                                    

The medium also contains 100 mg/liter ampicillin. The ampicillin isoptional for production but is always found in the medium used forgrowing the inoculum.

Biotin, thiamine and ampicillin in concentrated solution were filtersterilized separately and added to the sterile production medium beforeinoculation. Sterile gulucose solution was added initially to supply 10g/l. At the induction step another 10 g/l of glucose was added.

The trace elements solution contains

    ______________________________________                                                FeCl.sub.3     16     g/l                                                     ZnCl.sub.2 4H.sub.2 O                                                                        2      g/l                                                     CoCl.sub.2.6H.sub.2 O                                                                        2      g/l                                                     Na.sub.2 MoO.sub.4.2H.sub.2 O                                                                2      g/l                                                     CaCl.sub.2.2H.sub.2 O                                                                        1      g/l                                                     CuCl.sub.2     1      g/l                                                     H.sub.3 BO.sub.3                                                                             0.5    g/l                                                     Conc. HCl      100    ml/l                                            ______________________________________                                    

The medium is inoculated with 0.5-10% inoculum culture and incubated at30° C. Agitation-aeration rates are set to maintain a dissolved oxygenlevel above 20% air saturation. The pH is maintained at 7±0.2 with NH₃.Once cell concentration reaches about 3.5 g/l (OD₆₆₀ =10) induction isstarted.

The temperature is raised to 42° C. and maintained at 42° C. for 1-5hours. The culture is then chilled and cells are recovered bycentrifugation for hormone purification.

Recovery of bGH

Thirteen kilograms of bacterial cells (wet cake) are resuspended in 5volumes of a solution containing 50 mM sodium phosphate buffer (pH 7.4),50 mM EDTA and 100 mM NaCl, using a Polytron (Kinematica) blender, whilecontrolling the blender's speed to minimize foaming. The homogenoussuspension is continuously passed through a Dynomill cell disruptor KD5(Willy A. Bachofen, Basel) at a rate of 80 liter per hour and thehomogeneous suspension of disrupted cells clarified by centrifugation ina CEPA 101 centrifuge at a flow rate of 45 liter per hour. Theprecipitate from the centrifugation step is collected and resuspended in15.5 liters of 50 mM sodium phosphate buffer (pH 7.4) containing 50 mMEDTA. nysoszyme is added to a final concentration of 0.05 mg/ml and thesuspension incubated for 16 hours at 37° C. Triton X-100 is added to afinal concentration of 1%. The suspension is then incubated for 30minutes at room temperature, sonicated in a continuous flow cellsonificator (Heat System) at a rate of 18 liters per hour andcentrifuged in a CEPA 101 centrifuge. The precipitate is collected,resuspended in 50 mM sodium phosphate buffer (pH 7.4), sonicated asabove, and centrifuged in a CEPA 101 centrifuge. The cells areresuspended in 15.5 liters of 50 mM sodium phosphate buffer (pH 7.4)containing 50 mM EDTA and 100 mM NaCl and twice precipitated andresuspended in 15.5 liters of distilled water. The precipitate iscollected by centrifdgation and stored at -20° C.

Purification of bGH

The precipitate is resuspended in 30-40 liters distilled water andsolubilized by titration with 0.5N NaOH to pH 11.8. The solution is thencontinuously sonicated and clarified by centrifugation in CEPA 101centrifuge if necessary, or filtered through Whatman No. 1 paper.

The clarified protein solution (32.6 liters containing 297,000 OD's at280 nm) is divided into separate portions (6×5.4 liters) each containing50,000-60,000 OD's. Each portion is ultrafiltered separately through aMillipore Pellicon ultrafilter equipped with three 100,000 molecularweight cutoff cassettes (type PTHK) of 5 ft² area each. A 5.4 literportion is concentrated to 1 liter retentate volume. The ultrafiltrateis collected and saved. The retentate is diluted back to its originalvolume with fresh 10 mM Borate buffer, pH 11.8, and mixed well. Thebatch is concentrated again to 1 liter retentate volume. Theultrafiltrate is collected and combined with the first ultrafiltrate.When the running total of the OD's in the ultrafiltrates equals 20% ofthe OD's initially charged to the ultrafilter, the retentate volume onthe next concentration step is taken to 0.5 liters instead of 1 liter.The cycle of concentration and dilution with 10 mM Borate buffer iscontinued until the ultrafiltrate from a retentate volume of 0.5 litershas an absorbance at 280 nm (1 cm cell) of less than 0.1. This normallytakes between 9 and 12 cycles of concentration and dilution. The finalretentate is discarded.

All ultrafiltrates are combined and adjusted to pH 9.0 with 6N HCl. Theother 5.4-liter portions are ultrafiltered in the same fashion, and allpH adjusted ultrafiltrates are combined. A typical run produces a totalof 380 liters of ultrafiltrates with an absorbance of 0.26 equivalent to100,000 OD's and requires 24 to 40 hours to complete. The combinedultrafiltrates (380 liters containing 100,000 OD's at 280 nm) from the100K ultrafiltration step are loaded onto a Sepharose CL-6B DEAEion-exchange column at a linear flow velocity of 23 cm/hr (25 liter/hr).The 37-cm diameter 15-cm high column is washed with two bed volumes (32L) of 10 mM Borate buffer at pH 9.0. The eluate from the loading andwashing steps is discarded. A Step change in eluent to 10 mM Borate, 100mM sodium chloride, pH 9, displaces the bGH off the column. The elutionflow velocity is 23 cm/hr. The progress of the run is monitored byfollowing absorbance of the eluate at 280 nm. The bGH. peak is collectedin 4 to 5 bed volumes (84 liters containing 43,000 OD's at 280 nm) andthen concentrated to approximately 10 mg/ml using a Millipore Pelliconultrafiltration device with a 10,000 molecular weight cutoff cassettes.The solution is then lyophilized. The yield is approximately 70 g ofpure bGH.

EXAMPLE 6 Activity Of bGH Analog Produced By pRO12 RadioimmunoassayComparison of bGH Analog with Natural bGH

A solution containing 100 ng/ml bGH analog was prepared in phosphatebuffered saline (1% BSA). This solution was diluted serially toconcentrations of 50, 25, 12.5, 6.25, 3.12, 1.56 and 0.78 ng/1.Duplicate 0.1 ml aliquots of these solutions were submitted to RIA usinga double antibody procedure. The dilution curve was comparable to thatobtained with natural bGH.

2. Radioreceptor Binding Assay

A radioreceptor binding assay was performed with rabbit liver membranesas described by Tushima, T. and Freisen, H. G., (Y. Chin., Endocr.Metab. (1973), 37, 3) using 125_(I) -construction bGH as the tracer andauthentic bGH solutions for the construction of calibration curves.Samples were incubated in triplicate for two hours at room temperaturein 0.3 ml of assay buffer (50 mM Tris, 15 mM CaCl₂ and 5 mg/ml bovineserum albumin, pH 7.6). The tubes contained ¹²⁵ I-bGH (20,000 cpm ofpreparation of 30-60 μci/μg), 150-250 μg liver membrane protein andeither natural bGH (1-100 ng) or extracts of bacterial bGH. The resultdemonstratred that the bGH activity of the bGH analog is comparable tothat of natural bGH.

3. Tibia Test

The bioactivity of the pRO12 produced bGH analog recovered frombacterial cells according to Example 5 was evaluated by a tibia test.(Parlow, A.F., et al., Endocrinology (1965) 77, 1126). Rats werehypophysectomized at 28-30 days of age, then kept for 10-14 days withouttreatment. Bovine growth hormone derived from bovine pituitaries or fromrecombinant Escherichia coli was dissolved in 0.15M NaCl+0.01M borate,pH 10.0. Rats (4-7 per group) received daily subcutaneous injections ofbGH solutions (5-125 μg/day in 0.2 cc) for 5 days while kept on a normaldiet (Purina Rat-Chow and water adlibitum). The animals were sacrificedon the 6th day, their foreleg knee-bones taken out, cut longitudinally,fixed with acetone and stained with 2% AgNO₃. The width of theepiphyseal plates was measured by observation through a dissectingbinocular (Nikon). Mean values (40 readings per rat) were used for theconstructon of long dose-response curves. The results demonstrated thatthe bGM activity of the pRO12-produced bGH analog is comparable to thatof natural bGH.

EXAMPLE 7 Growth Of SODβ₁ T₁₁ 1. Stock Cultures

Stock cultures of pSODβ₁ T₁₁ were grown on casein medium (see Inoculum),then diluted two-fold with freezing medium and stored at -80° C.Freezing medium contains per 500 ml:

    ______________________________________                                        K.sub.2 HPO.sub.4     6.3    gr                                               KH.sub.2 PO.sub.4     1.8    br                                               Na Citrate            0.45   gr                                               MgSO.sub.4.7H.sub.2 O 0.09   gr                                               (NH.sub.4).sub.2 SO.sub.4                                                                           0.9    gr                                               Glycerol              44.0   gr                                               ______________________________________                                    

The inoculum was propagated in 20 g/l casein hydrolysate, 10 g/l yeastextract and 2 g/l NaCl. Sterile medium in a shake fiask was inoculatedfrom stock culture and incubated 15 hours on a shaker at 30° C. andapproximately 200 r.p.m. As needed subsequent stages in inoculumpropagation were carried out in stirred aerated fermenters. Sterilemedium was innoculated with 2-10% innoculum and incubated 15 hours at30° C., pH 7±0.5 with agitation and aeration to maintain a dissolvedoxygen level adore 20% air saturation.

III. Production

The production medium contains:

    ______________________________________                                        Casein hydrolystae     20     g/l                                             Yeast extract          10     g/l                                             K.sub.2 HPO.sub.4      2.5    g/l                                             MgSO.sub.4./7H.sub.2 O 1      g/l                                             NaCl                   5      g/l                                             Biotin                 0.1    mg/l                                            Thiamine               1      mg/l                                            Trace elements solution                                                                              3      ml/l                                            CuSO.sub.4             0.8    g/l                                             ZnSO.sub.4             10     mg/l                                            ______________________________________                                    

The medium also contains 12.5 mg/liter tetracycline. The tetracycline isoptional for production, but is always found in the medium used forgrowing the inoculum.

Biotin, thiamine and tetracycline in concentrated solution were filtersterilized separately and added to the sterile production medium beforeinoculation. Sterile glucose solution was added initially to supply 10g/l. At the induction step another 10 g/l of glucose was added.

The trace elements solution contains:

    ______________________________________                                        FeCl.sub.3           16     g/l                                               ZnCl.sub.2.4H.sub.2 O                                                                              2      g/l                                               CoCl.sub.2.6H.sub.2 O                                                                              2      gf/l                                              Na.sub.2 MoO.sub.4.2H.sub.2 O                                                                      2      g/l                                               CaCl.sub.2.2H.sub.2 O                                                                              1      g/l                                               CuCl.sub.2           1      g/l                                               H.sub.3 BO.sub.3     0.5    g/l                                               Conc. HCl            100    ml/l                                              ______________________________________                                    

The medium is inoculated with 0.5-10% inoculum culture and incubated at30° C. Agitation-aeration rates are set to maintain a dissolved oxygenlevel above 20% air saturation. The pH is maintained at 7±0.2 with NH₃.Once cell concentration reaches about 3.5 g/l (OD₆₆₀ =10) induction isstarted.

The temperature is raised to 42° C. and maintained at 42° C. for 1-5hours. The culture is then chilled and cells are recovered bycentrifugation for enzyme Purification.

Recovery Of SOD

One and half kilograms of bacterlal ceils (wet cake) are suspended in 12liters of 50 mM sodium phosphate (pH 7.8), in a Polytron (Kinematica)blender while controlling the speed to minimize foaming. The homogeneoussuspension is continuously passed through a Dynomill cell disrupter KD5(Willy, A. Bachofen, Basel). The homogeneous suspension of disruptedcells is sonicated using a continuous flow cell and centrifuged in aCEPA 101 centrifuge. The supernatant is heated for 2 hours at 65° C.,cooled and centrifuged as before. The clear supernatant is concentratedto 1 liter in a Millipore Pellicon ultrafiltration device using 10,000molecular weight cutoff cassettes (type PTGC). The concentrated proteinsolution is passed through a DEAE-Sepharose column (2 Kg DEAE Sepharose)equilibrated with 150 mM sodium phosphate buffer (pH 7.8). The flowthrough solution is collected, concentrated and dialyzed in a Pelliconultrafiltration device against 20 mM Tris-HCl, pH 7.8, and then appliedon to a QAE-Sepharose column equilibrated with 20 mM Tris-HCl buffer.The column is developed with a 20 mM Tris HCl buffer, pH 7.8, and a saltgradient (0-200 mM NaCl). SOD-containing fractions are collected,concentrated using a Pellicon ultrafiltration device, dialzed againstdistilied water and then brought to 100 mM sodium acetate by adding 1Msodium acetate buffer, pH 4.8. The protein solution is then furtherseparated on a CM-Sepharose column equilibrated with 100 mM sodiumacetate buffer, pH 4.7. The column is developed using the same bufferand a salt gradient (100-500 mM NaCl). SOD containing fractions arecoliected, concentrated using a Pellicon ultrafilter device andlyophilized.

EXAMPLE 8 Activity Of SOD Produced By pSODβ₁ T₁₁

The enzymatic activity of the SOD analog produced by pSODβ₁ T₁₁-prepared in Example 7 was assayed by monitoring the inhibition ofreduction of ferricytochrome-c as described by McCord and Fridovich, J.Biol. Chem. (1969), 244:6049-6055. The results demonstrated that theactivity of pSODβ₁ T₁₁ -produced SOD analog was comparable to that ofnatural human SOD (Sigma) and to that of bovine SOD (Orgotein:Grunenthal GMBH).

EXAMPLE 9

The yield and activity of human superoxide dismutase produced by the SODhost-vector systems described in Example 3 may be improved by modifyingthe growth conditions of the host-vector systems. As the following datademonstrates supplementing the growth medium for the host-vector systemwith Cu and Zn results in a greater yield of the enzyme in active dimerform.

Growth of Bacteria Containing pSODβ₁ T11 I. Stock Cultures

Stock cultures of pSOD.sub.β1 T11 were grown on casein medium (seeinoculum), then diluted twofold with freezing medium and stored at -80°C. Freezing medium contains:

    ______________________________________                                        K.sub.2 HPO.sub.4     6.3    gr                                               KH.sub.2 PO.sub.4     1.8    gr                                               Na Citrate            0.45   gr                                               MgSO.sub.4.7H.sub.2 O 0.09   gr                                               (NH.sub.4).sub.2 SO.sub.4                                                                           0.9    gr                                               Glycerol              44     gr                                               Per 500               ml                                                      ______________________________________                                    

II. Inoculum

The inoculum was propagated in 20 g/l casein hydrolysate, 10 g/l yeastextract and 2 g/lNaCl. Sterile medium in a shake flask was inoculatedfrom stock culture and incubated 15 hours on a shaker at 30° C., andapproximately 200 r.p.m. If needed subsequent stages in inoculumpropagation were carried out in stirred aerated fermenters. Sterilemedium was inoculated with 2-10% flask culture, and incubated 15 hoursat 30° C., pH 7±0.5 with agitation and aeration to maintain dissolvedoxygen level above 20% air saturation.

III. Production

The production medium contains:

    ______________________________________                                        Casein hydrolystae     20     g/l                                             Yeast extract          10     g/l                                             K.sub.2 HPO.sub.4      2.5    g/l                                             MgSO.sub.4.7H.sub.2 O  1      g/l                                             NaCl                   5      g/l                                             Biotin                 0.1    mg/l                                            Thiamine               1      mg/l                                            Trace elements solution                                                                              3      ml/l                                            Tetracycline           12.5   mg/l                                            ______________________________________                                    

In some of the experiments we added:

    ______________________________________                                        CuSO.sub.4.5H.sub.2 O 0.8   g/l                                               ZnSO.sub.4.7H.sub.2 O 10    mg/l                                              ______________________________________                                    

Biotin, thiamine, and tetracycline in concentrated solutions were filtersterilized separately and added to the sterile production medium beforeinoculation. Sterile glucose solution was added initially to supply 10g/l. At the induction step another 10 g/l of glucose was added.

The trace elements solution contains:

    ______________________________________                                        FeCl.sub.3            16     g/l                                              ZnCl.sub.2.4H.sub.2 O 2      g/l                                              CoCl.sub.2.6H.sub.2 O 2      g/l                                              Na.sub.2 MoO.sub.4.2H.sub.2 O                                                                       2      g/l                                              CaCl.sub.2.2H.sub.2 O 1      g/l                                              CuCl.sub.2            1      g/l                                              H.sub.3 BO.sub.3      0.5    g/l                                              Conc. HCl             100    ml/l                                             ______________________________________                                    

The medium is inoculated with 0.3-10% inoculum culture and incubated at30° C. Agitation-aeration rates are set to maintain dissolved oxygenlevel above 20% air saturation. The pH is maintained at 7±0.2 with NH₃.Once cell concentration reaches about 3.5 g/l (OD₆₆₀ =10) induction isstarted.

The temperature is raised to 42° C. and maintained at 42° C. for 1-5hours. The culture is then chilled, and cells are recovered bycentrifugation for enzyme purification.

RECOVERY OF SOD

One and one-half kilograms of bacterial cells (wet cake) are suspendedin 12 liters of 50 mM sodium phosphate (pH 7.8), in a Polytron(Kinematica) blender while controlling the speed to minimize foaming.The homogeneous suspension is continuously passed through a Dynomillcell disrupter KD5 (Willy, A. Bachofen, Basel). The homogeneoussuspension of disrupted cells is sonicated using a continuous flow celland centrifuged in a CEPA 101 centrifuge. The supernatant is heated for2 hours at 65° C., cooled and centrifuged as before. The clearsupernatant is concentrated to 1 liter in a Millipore Pelliconultrafiltration device using 10,000 molecular weight cutoff casettes(type PTGC). The concentrated protein solution is passed through aDEAE-Sephacel column (2 Kg DEAE Sephacel) equilibrated with 150 mMsodium phosphate buffer (pH 7.8). The flow through solution iscollected, concentrated and dialyzed in a Pellicon ultrafiltrationdevice against 20 mM Tris-HCL, pH, 7.8 and then applied on to aQAE-Sepharose column equilibrated with 20 mM Tris-HCl buffer. The columnis developed with a 20 mM Tris-HCl buffer, pH 7.8, and a salt gradient(0-200 mM NaCl). SOD containing fractions are collected, concentratedusing a Pellicon ultrafiltration device, dialyzed against distilledwater and then brought to 100 mM sodium acetate by adding 1M sodiumacetate buffer, pH 4.8. The protein solution is then further separatedon a CM-Sepharose column equilibrated with 100 mM sodium acetate buffer,pH 4.7. The column is developed using the same buffer and a saltgradient (100-500 mM NaCl). SOD containing fractions are collected,concentrated using a Pellicon ultrafiltration device and lyophilized.

EXAMPLE 10 Reduction in Reperfusion Injury with Recombinant HumanSuperoxide Dismutase Following Global Ischemia

Human superoxide dismutase produced by the host-vector systempSOD.sub.β1 Tll in E. coli A1645 described in Example 3, grown andpurified under the conditions described in Example 9 has been shown toreduce reperfusion injury following global ischemia.

Isolated Perfused Rabbit Heart Preparation

Female New Zealand white rabbits, 1.2-2.0 kg were heparinized andanesthetized, their hearts removed and quickly placed into cold (4° C.)perfusate. The ascending aorta was cannulated and the hearts perfusedunder constant pressure (110 cm of water) with a modified Krebs-Ringersbicarbonate buffer solution containing 117 mM sodium chloride, 6 mMpotassium chloride, 3.0 mM calcium chloride, 1.0 mM magnesium sulphate,0.5 mM EDTA, and 16.7 mM glucose with the final pH adjusted to 7.40 bythe addition of approximately 24 mM sodium bicarbonate. The perfusatewas bubbled continuously with 95% oxygen and 5% carbon dioxide. Coronaryflow was removed from the NMR sample tube by vacuum aspiration. Thehearts were paced at 175 beats/minute by right ventricular pacing with awick soaked in saturated potassium chloride, encased in polyethylenetubing, and connected to a Grass SD-9 stimulator. To quantitate leftventricular contractile function a latex rubber balloon was tied to theend of a 100 cm length of PE 190 tubing, carefully purged of air bubblesand connected via a threeway stopcock to a Statham P23Db transducer.Isovolumic pressure was recorded with a Brush two channel direct writingrecorder. The balloon was initially inflated via a syringe with a volumeof saline sufficient to produce an end diastolic pressure of 10 mmHg.All subsequent measurements of developed pressure were at this enddiastolic volume. All hearts were subjected to 30 minutes of globalischemia during which time the hearts were maintained at 37° C. by aflow of warm perfusate around the heart. Total interruption of aorticin-flow was accomplished by cross clamping the perfusion line.Forty-five minutes of normothermic reperfusion (37°±2° C.) followed theperiod of ischemia. Recovery of left ventricular developed pressure wascalculated as a percentage of the pre-ischemic control. At the onset ofischemia the balloon was deflated and the pacer turned off. The balloonwas reinflared 15 minutes after initiating reflow, just prior to thefirst measurement of function with the same volume removed at the onsetof ischemia. Coronary blood flow was measured volumetrically by vacuumaspiration prior to ischemia, 5 minutes after reflow and after 15, 30and 45 minutes of reperfusion.

Nuclear Magnetic Resonance Methods

Phosphorous-31 NMR spectra were obtained in a Brucker WH 180spectrometer at 4.23 Telsa in a wide bore superconducting magnet. Atthis field strength, phosphorus resonates at 72.89 MHz. The diameter ofthe phosphorus probe is 25 mm. This instrument was operated in thepulsed Fourier transform mode and interfaced to a Nocolet 1280 computerand the data collected on high density magnetic disks. Because of thefield stability of the superconducting magnet, field/frequency lock isnot required. Five minute proton alecoupled spectra were collected fromtransients following 45° pulses delivered at two second intervals,conditions previously documented to result in minimal spectralsaturation. The data were accumulated with a 2K table at a 3,000 Hzspectral width.

Estimation of Tissue Intracellular pH from NMR Spectra

Measurement of intracellular pH was determined from the chemical shift(δ_(o)) of the inorganic phosphate peak by the following equation:##EQU1##

In order to minimize tissue inhomogeneity effects, chemical shift valueswere measured relative to the resonance of phosphocreatine which isrelatively pH independent over the range of pH to be encountered inthese studies (pK_(A) =4.6). The constants used in this equation arepK=6.90, δ_(A) =3.290 PPM and δ_(B) =5.805 PPM, as previously reported.

Quantitation of Metabolites from NMR Spectra

Estimations of tissue phosphocreatine (PCr), adenosine triphosphate(ATP), as well as inorganic phosphate (Pi) were obtained by planimetricmeasurements of the areas under the individual peaks allowing for thecomputer determined normalization constant or scaling factor. AHewlett-Packard digitizer was used to perform the area integrations.Quantitative data thus derived for PCr, ATP and Pi are expressed aspercent of the pre-ischemia control content.

Experimental Protocol

Seventeen hearts were divided into two groups:

Group I (n=8) The hearts in this group were treated with 60,000 units ofhuman recombinant superoxide dismutase (hSOD, specific activity 3,200IU/mg) administered as a 10 ml bolus just prior to reflow, followed by acontinuous infusion of 60,000 units during the first 15 minutes ofreflow; hSOD was dissolved in warm 37° C. Krebs-Ringers bicarbonateperfusate.

Group II (n=9) The hearts in this group received a 10 ml bolus ofperfusate just prior to reflow followed by normothermic reperfusion.

Results Recovery of Left Ventricular Function

The experimental ischemic model employed in the present study wasspecifically chosen following a series of preliminary studies to providehearts which having suffered a moderately severe irreversible insult,still retained the potential for improvement with a therapeuticintervention. At the end of 45 minutes of reflow, the recovery of leftventricular function (as measured by percent recovery of controldeveloped pressure) was 47±5% for the control group with an enddiastolic pressure to 48±7 mmHg (compared to a pre-ischemic controlvalue of 10 mmHg). These parameters did not change appreciably between30 and 45 minutes of reperfusion, suggesting that a relatively steadystate of recovery had been achieved. The administration of hSOD justprior to fellow and for the initial 15 minutes of reperfusion resultedin significantly improved preservation of cardiac function and in asmaller increase in end diastolic pressure compared to control hearts;hSOD treated hearts recovered 71±6% of control developed pressure at anend diastolic pressure of only 27±4 mmHg (both P 0.01 vs control).

Myocardial Metabolism During Ischemia and Following Reperfusion

Myocardial high-energy phosphate contents were serially measured duringischemia and following reperfusion in both control and hSOD-treatedhearts. During the 30-minute ischemic period progressive decreases inmyocardial creatine phosphate and ATP content were observed.Phosphocreatine content fell by the end of the ischemic period to 8±3%of the pre-ischemic baseline value in control hearts and to 10±5% ofcontrol in those hearts which were subsequently to be treated with hSOD;ATP content reached 36±6% of the baseline value in control hearts and33±6% in hSOD treated hearts. These data clearly indicate that bothgroups of hearts were subjected to an equally severe degree of ischemia.These data also rule out the possibility the hearts receiving hSODdemonstrated better functional recovery due to better preservation ofcellular metabolism during the ischemic period by some uncontrolled formechanism.

At the end of 45 minute reflow period hSOD-treated hearts displayed anearly normal content of phosphocreatine (93±9% of control) whereascontrol hearts recovered only 69±7% of the original value (P <0.05). Atthe end of the reflow period ATP content was equal in both groups ofhearts (41±4% in control vs 42±5% in hSOD-treated hearts). This latterresult might reflect increased energy demands hearts recovering betterleft ventricular function, in the presence of a limited ability toincrease rates of high energy phosphate production. In contrast, poorerrecovery of function in control hearts would result in less utilizationof high energy phosphate metabolites, possibly masking even more severelimitation in energy production.

In conclusion, these data demonstrate, in hearts subjected to amoderately severe ischemic insult and subsequent reperfusion, theadministration of hSOD just prior to and during early reperfusionresults in better recovery of systolic and diastolic function, as wellas in higher myocardial content of phosphocreatine. These data alsosuggest that reperfusion of ischemic myocardium may result in acomponent of structural and/or functional damage which can be avoided orreduced by the administration of an oxygen free radical scavenger suchas hSOD at the time of reperfusion. Thus, by limiting that component ofreflow injury resulting from reoxygenation of previously ischemicmyocardium, hSOD may provide a valuable addition to thrombolytic therapyand/or coronary angioplasty in patients treated early after acutemyocardial infarction.

EXAMPLE 11 Reduction in Experimental Infarct Size by Recombinant HumanSuperoxide Dismutase Administration During Reperfusion

Human superoxide dismutase produced by the host vector system pSOD β₁Tll in E. coli A1645 described in Example 3, grown and purified underthe conditions described in Example 9 has been shown to reduce infarctsize in hearts.

Timely reperfusion of the ischemic myocardium reduces infarct size (IS);however, this beneficial effect may be blunted by the simultaneousoccurrence of reflow injury, mediated through the generation of toxicoxygen free radicals. To test whether scavenging of free radicals byrecombinant human superoxide dismutase (hSOD) could result in areduction of IS compared to reperfusion alone, in 16 anesthetized dogsthe circumflex coronary artery was occluded before any marginal branchfor 90 min; at the time of reperfusion the animals were injected witheither hSOD (400,000 units as a bolus into the left atrium, followed by300,000 units as a 1 hr i.v. infusion; n=8), or with a similar amount ofsaline (controls, n=8). The chest was then closed and the animals wereallowed to recover. After 48 hrs the dogs were sacrificed and the heartsprocessed in a blinded fashion for the evaluation of IS by grosspathology and of the risk area by postmortem angiography. Proximalocclusion of the circumflex artery resulted in ischemia of 40.8+2.3% ofthe left ventricle (LV) in controls and 41.8+2.0 in treated dogs. Incontrol dogs reperfusion was associated with infarction of 52.2+7% ofrisk area; hSOD treatment, however, resulted in a significant reductionof necrosis, IS being 33.6+2.1% of risk area (p<0.05). Control animalsdeveloped confluent, non-transmural infarcts which extended throughoutmost of the risk area, whereas in treated dogs the infarcts appearedmore patchy and non-confluent. In conclusion, free radical scavenging byhSOD administered at the time of reperfusion significantly reduced theextent of necrosis, possibly through a prevention of reflow injury.

EXAMPLE 12 The Role of Oxygen Free Radicals in Mediating the ReperfusionInjury of Cold Preserved Ischemic Kidneys¹

In a new indication, human superoxide dismutase can reduce reperfusioninjury following transplantation of organs. The following Exampledemonstrates that superoxide dismutase ameliorates injury on reperfusionfollowing the transplantation of a kidney. The human superoxidedismutase utilized in this example was produced by the host-vectorsystem pSODβ₁ Tll in E. coli A1645 described in Example 3, and grown andpurified under the conditions described in Example 9.

The parenthesized arabic numbers found throughout this Example refer tothe articles listed in the Bibliography at the end of this Example.

Model of Renal Preservation and Transplantation Ischemia in Swine

Female, outbred pigs, weighing 15 to 18 kg were premedicated withacepromazine and attopine, and anesthetized with ketamine and halothane.In the donor pigs, diuresis was established 30 minutes prior to harvestby the intravenous administration of 1500 cc of Ringer's lactate,furosemide (20 mg) and mannitol (12.5 g). Phenoxybenzamine (50 mg) wasgiven intravenously to prevent renal vasopasm, which can be seenfrequently in pigs. Through a midline abdominal incision, the distalaorta and vend cava were mobilized, just proximal to their bifurcations.The ureters were dissected and divided at the level of the bladder. Aninflow catheter was placed in the aorta, just above the bifurcation, andan outflow catheter was placed into the inferior vena cava. Heparin(5,000 units) was given intravenously and a continuous flush withEuro-Collins solution at 4° C. was initiated through the distal aorta,coincident with the cross-clamping of the aorta just above the renalartery. After the kidneys had been cooled in situ, they were removed enblock. The kidneys were then separated and one was assigned to be thecontrol and the other to be the test kidney, thus facilitating a paireddesign. Each kidney was then flushed again with 4° C. Euro-Collinssolution. When called for by the protocol, test substances were added tothe preservation fluid of the second kidney at this time. Both kidneyswere packaged sterilely and stored at 4° C. overnight.

After 24 hrs of cold ischemia, a fresh recipient animal was anesthetizedand the preserved kidneys were transplanted to its iliac vessels. Thetime required for each anastomosis was 25 to 30 min. the control(untreated) kidney was always transplanted first, the test kidneysecond. Reperfusion of the test kidney was therefore always delayed fora total of one hour after the reperfusion of the control kidney. Thismeans that the control kidneys were subjected to 23 hours of coldischemia, the test kidneys to 24 hours. Test drugs were administeredsystemically (allopurinol) or intraarterially (superoxide dismutase=SOD)beginning one hour after reperfusion of the control kidney at the timeof reperfusion of the test kidney. Thus, the control kidney was exposedto the toxic effects of rewarming and reperfusion for one hour beforeexposure to any possible effects of agents provided to the test kidney.Following reperfusion of the second kidney, the native kidneys of therecipient pig were removed. Additional doses (10 g) of mannitol weregiven prior to reperfusion of first the control, and then again of thetest kidney, to mimic clinical practice. This allowed evaluation of theeffect of free radical modifying agents superimposed upon optimalconventional preservation/transplantation tecnniques. The ureter fromeach kidney was brought out separately as a cutaneous ureterostomy.

Two days following transplantation, the recipient was lightlyanesthetized and urine was collected for one hour from each kidney(ureterostomy) separately and assayed for volume and creatinineconcentration. Serum creatinine was also determined, allowing thecalculation of creatinine clearance separately for each kidney.

All results are expressed as mean±SEM. Data were analyzed during theStudent t-test (two-tailed). In most cases a paired test could beapplied due to the paired design of the experiments.

Experimental Protocol Superoxide Dismutase (SOD) and Catalase

Sigma bovine blood superoxide dismutase, a scavenger of oxygen freeradicals, was administered to test kidneys in four pigs. A 5 mg boluswas given into the renal artery immediately prior to reperfusion, and aconstant intra-arterial infusion was maintained at 1 mg/min during thefirst 15 min. of reperfusion. This provided a total dose of 20 mg ofSOD. In a second group of four pigs, the test kidneys received catalase(Sigma Co.), by the same dosage regimen, in addition to SOD. The otherkidney in each of these pigs received no treatment, and thus served as acontrol.

Dose Response to SOD

In order to determine the minimal dosage for maximal protection, a doseresponse relationship was studied. At revascularization, one kidneyreceived an infusion of a lesser of two doses of SOD and the other thenext higher dose. Human recombinant SOD prepared as described in Example9 was given as described above. Two comparisons were made at each dosagerange. In the first, 0.2 mg of SOD was compared with saline solution asthe control. Stepwise, 0.2 mg was compared to 2 mg, 2 mg was compared to20 mg, and finally 20 mg to 100 mg. In each case, the kidney to receivethe lesser dose was transplanted first, in order to avoid the possibleproblem of SOD retention in the circulation at the time of the secondtransplant.

Results The Effect of Bovine SOD and Catalase

The creatinine clearance for a normal single kidney in pigs of the sizewe used, under the above conditions of anesthesia and hydration, was25.5+6.3 ml/min (n=8). The administration of SOD in a dose of 20 mg intothe renal artery for the first 15 min of reperfusion substantiallyameliorated the renal functional impairment after cold ischemia. Thefour kidneys treated with SOD alone had a mean creatinine clearance of23.2±4.5 ml/min), almost three times that of the control kidneys(8.4±1.7 ml/min, p<0.05). A combination of SOD and catalase providedsimilar, but not greater, protection (C_(CR) =19.0±4.5 ml/min). In earlypilot experiments, a separate group of four pigs had undergonetransplantation with anastomotic times in excess of 40 minutes. Althoughrenal function was significantly increased by SOD and catalase, in theseanimals, both treated and control kidneys had very poor function (C_(CR)=4.8±0.8 vs. 1.6±0.4 ml/min ). In these kidneys, presumably subjected tomore severe injury due to ischemia per se prior to reperfusion,modification of free radical injury was unable to restore normal renalfunction.

Human SOD Analog Dose Response Relationships

Infusions of human SOD analog (0.2 mg and 2 mg) provided no improvementin renal function compared to the controls. However, the mean creatinineclearance of kidneys receiving 20 mg of SOD analog was 14.2+1.1 ml/min,significantly better than in the pairs receiving 2 mg infusions (7.7±1.0ml/min p<0.05). No further benefit was obtained from 100 mg of SODanalog (C_(CR) =16.1±1.2 ml/min). Therefore, the minimal effective doseof SOD analog proved to be greater than 2 and less than 20 mg whenadministered in this manner. The human SOD analog was as effective asthe bovine SOD.

Discussion

The paired design was employed to maximize differences due to thetreatment regimens employed, and to control for confounding variableswhich were related to the particular donor or recipient animal. It alsoprovided for a paired statistical analysis of results, which allowedoptimal use of small numbers of relatively expensive experimentalanimals.

The striking finding of these studies was the magnitude of the benefitprovided by ablation of free-radical mediated reperfusion injury.Despite the fact that the control kidneys received the benefits ofoptimal conventional methods for organ preservation, including healthydonor kidneys and recipient animals, hydration, alpha adrenergicblockage, anticoagulation, and diuresis, they demonstrated a severefunctional lesion, with creatinine clearance levels depressed to valuesless than a third of normal. This 24 hour period of ischemicpreservation, as it often does in similar clinical circumstances,exceeds conventional organ preservation capabilities. Treatment witheffective doses of bovine SOD or human SOD analog dramatically preventedthis injury, preserving renal function at near normal levels. Indeed,there was not a single kidney so treated that did not have a creatinineclearance at least twice that of its paired, untreated control, whenmeasured 48 hours after transplantation. The magnitude of this benefitwas therefore quantitatively greater than that seen by others following45-60 minutes of warm ischemia. This suggests that with optimalconventional preservation techniques, the injury due to ischemia per sehas been minimized, allowing the injury produced at reperfusion tobecome predominant. This interpretation is further supported by thestudies with kidneys preserved 18 hours prior to reperfusion. In thesekidneys, function was excellent in both the control and the treatmentgroups, suggesting that sufficient time had not elapsed to allow theaccumulation of enough matabolites to set up the conditions favoringfree radical generation at reperfusion. On the other hand, when thekidneys were subjected to a more severe degree of ischemia per se, as inthe early pilot experiments with prolonged anastomosis (i.e., warmischemia) times, SOD was not able to restore function to near normallevels, although a significant improvement was still seen. These kidneyswould therefore be more analogous to those studied following shorterperiods of warm ischemia (7,8,9). As in other organs, the benefits ofobviating free radical injury are primarily related to the relativeproportions of the injury that are due to ischemia itself, compared tothat due to reperfusion.

Furthermore, the achievement of this increment of benefit appears to beobtained either wholly, or not at all. The dose response studies withSOD showed either maximal protection, or no protection whatsoever.Catalase provided no additional benefit when added to SOD alone. Withinthe quantitative limits of resolution of this study, the prevention ofreperfusion injury appears to have been an all-or-nothing phenomenon.

The findings of this study, however, appear to be particularly relevantto clinical application. The 24 hour period of cold ischemia chosencorresponds well with the periods of cadaveric graft preservationnecessitated by clinical circumstances. Furthermore, these studiesevaluated the efficacy of free radical reperfusion injury ablation inthe face of optimal conventional preservation and transplantationmethods. This includes the use of mannitol, itself a potent hydroxylradical scavenger. These studies therefore suggest that a similar degreeof benefit might well be obtained by superimposing free radical ablationon current clinical practices. As SOD is a nontoxic compound, thisapproach seems promising.

BIBLIOGRAPHY

1. Parks D. A., Bulkley G. B., Granger D. N., Hamilton S. R., McCord J.M. Ischemic Injury to the Cat Small Intestine: Role of SuperoxideRadicals. Gastroenterology 82:9 (1982).

2. Manson P. N., Anthenelli R. M., Im M. J., Bulkley G. B., Hoopes J. E.The Role of Oxygen-Free Radicals in Ischemic Tissue in Island SkinFlaps. Ann Surg 198:87 (1983).

3. Shlafter M., Kane P. F., Kirsh M. M. Superoxide Dismutase PluseCatalase Enhance the Efficacy of Hypothermic Cardioplegia to Protect theGlobally Ischemic, Reperfused Heart. J Torac Cardiovasc Surg 83:830(1982).

4. Stuart R. S., Baumgartner W. A., Borkon A. M., et al. Five-HourHypothermic Lung Preservation with Oxygen Free-Radical Scavengers.Transplant Proc 17:1454 (1985).

5. Sanfey H., Bulkley G. B., Cameron J. L. The Role of Oxygen DerivedFree Radicals in the Pathogenesis of Acute Pancreatitis. Ann Surg200:405 (1984).

6. Hansson R., Gustavsson B., Jonsson O., et al. Effect of XanthineOxidase Inhibition on Renal

Circulation After Ischemia. Transplant Proc 14:51 (1982).

7. Ouriel K., Smedira N. G., Ricotta J. J. Protection of the KidneyAfter Temporary Ischemia: Free Radical Scavengers. J Vasc Surg 2:49(1985).

8. Peller M. S., Hoidal Jr, Ferris T. F. Oxygen Free Radicals inIschemic Acute Renal Failure in the Rat. J Clin Invest 74:1156 (1984).

9. Im M. J., Shen W. H., Pak C. I., Manson P. N., Bulkley G. B., HoopesJ. E. Effect of Allopurinol on the Survival of Hyperemic Island SkinFlaps. Plast Reconstr Surg 73:276 (1984).

10. Parks D. A., Bulkley G. B., Granger D. N. Role of Oxygen FreeRadicals in Shock, Ischemia, and Organ Preservation. Surgery 94:428(1983).

11. Toledo-pareyra L. H., Simmons R. L., Najarian J. S. Effect ofAllopurinol on the Preservation of Ischemic Kidneys Perfused with Plasmaor Plasma Substitutes. Ann Surg 180:780 (1974).

12. Vasco K. A., DeWall R. A., Riley A. M. Effect of Allopurinol inRenal Ischemia. Surgery 71:787 (1972).

13. Owens M. L., Lazarus H. M., Wolcott M. W., Maxwell J. G., Taylor J.B. Allopurinol nd Hypoxanthine Pretreatment of Canine Kidney Donors.Transplantation 17:424 (1974).

14. Granger D. N., Rutilli G., McCord J. Superoxide Radicals in FelineIntestinal Ischemia. Gastroenterology 81:22 (1981).

15. Roy R. S., McCord J. M. Superoxide and Ischemia: Conversion ofXanthine Dehydrogenease to Xanthine Oxidase. In: Greenwald R., Cohen G.,eds. Oxyradical and Their Scavenger Systems (Vol. 2). Cellular andMolecular Aspects. New York: Elsevier Science 145 (1983).

16. Toledo-pareyra L. H., Simmons R. L., Olson L. C., Najarian J. S.Clinical Effect of Allopurinol on Preserved Kidneys: A RandomizedDouble-Blind Study. Ann Surg 185:128 (1977).

17. Parks D. A., Granger D. N., Bulkley G. B. Superoxide Radicals andMucosal Lesions of the Ischemic Small Intestine (Abstract). Fed Proc41:1742 (1982).

18. Casale A. S., Bulkley G. B., Bulkley B. H., Flaherty J. T., Gott V.L., Gardner T. J. Oxygen Free-Radical Scavengers Protect the Arrested,Globally Ischemic Heart Upon Reperfusion. Surg Forum 34:313 (1983).

EXAMPLE 13 Survival of Isolated Rabbit Cornea and Free RadicalScavengers

Human superoxide dismutase produced by the host vector system pSODβ₁ Tllis E. coli A1645 described in Example 3, grown and purified under theconditions described in Example 9 was shown to prolong the survivalperiod of excised isolated corneas.

The parenthesized arabic numbers found throughout this Example refer tothe articles listed in the Bibliography at the end of this Example.

Previous studies established the beneficial effect of low concentrationof adenosine on the rabbit corneal endothelial pump in vitro (1).Activation of the fluid pump was obtained by perfusing the isolatedcornea with physiological concentration of glucose (5 mM) and adenosine(1-6 M) in a balanced salt solution; the survival time was about 7hours.

Our next goal was to increase the survival time. Superoxide dismutase(SOD) (2), scavenges the superoxide free radicals by catalyzing thereaction 0₂.sup..- +0₂.sup..- +2H⁺ →H₂ O₂ +O₂. Administration of SOD isknown to significantly increase the survival of ischemic tissues afterpartial anoxia (3).

Substantial degree of tissue damage resulting from ischemia occursduring the period of reperfusion and reoxygenation of the isolatedtissues. Most of that injury is mediated through the superoxide radicaland its descendants and may be prevented if superoxide dismutase (SOD)or catalase are able to prolong the survival of the isolated cornea.

We isolated corneas from albino rabbit weighing 2.0-3.0 kg. The detailsof the techniques used have been previously described (1). Briefly, theeyes were excised and the corneal epithelia scraped off; then thecorneas were isolated as described, and subjected to perfusion.

Results

The addition of 2 μg/ml of SOD analog to the Basal Salt solutioncontaining 5 mM glucose and 1 μM adenosine prolonged the survival timeof the isolated cornea from 7 hours to 14 hours. If the adenosine waseliminated, the survival time was prolonged to only 12 hours.

In summary, the SOD analog was demonstrated to be useful in prolongingthe survival time of isolated corneas. The SOD analog may be importantin prolonging the survival of other isolated organs as well. Similardata using bovine SOD has been published in Experimental Eye Research4:153-154 (1985).

BIBLIOGRAPHY

1. Neuwirth, O. and Dikstein, S. (1983). The effect of cyclic AMP on therabbit corneal endothelial fluid pump. Current Eye Res. 2(8), 565-567.

2. Fridovich, I. (1975). Superoxide dismutases. Ann. Rev. Biochem. 44,147-159.

3. Manson, P. N., Robert, M., Anthenelli, M. M., Michael, J., Bulkley,G. B. and Hoopes, J. E. (1983). The role of oxygen-free radicals inischemic tissue injury in Island skin flaps. Ann. Surg. 198, 87-90.

4. Michaelson, A. M. and Puget, K. (1980). Cell penetration by exogenoussuperoxide dismutase. Acta Physiol. Scand. Suppl. 492, 67-80.

5. Perlman, M. and Baum, J. L. (1974). The mass culture of rabbitcorneal endothelial cells. Arch. Ophthalmol. 92, 235-237.

6. Klyce, S. and Maurice, D. M. (1976). Automatic recording of cornealthickness in vitro. Invest. Ophthalmol. 15, 550-553.

7. Neuwirth Lux, O. (1984). Survival of rabbit corneal endothelial pump.Ph.D. thesis, submitted to the Senate of the Hebrew University ofJerusalem.

8. Baret, A. and Emerit, I. (1983). Variation of superoxide dismutaselevels in fetal calf serum. Mutation Res. 121, 293-297.

9. Marklund, S., Holme, E. and Hellner, L. (1982). Superoxide dismutasein extracellular fluids. Clin. Chim. Acta 126, 41-51.

10. Packer, J. E., Mahood, J. S., Mora Arellano, V. D., Slater, T. F.,Willson, R. L. and Wolgenden, B. S. (1981). Free radicals and singletoxygen scavengers: reaction of a peroxy radical with β-carotene,Diphenyl Furan and 1,4,Diazobicyclo(2,2,2)-octane. Biochem. Biophys.Res. Commun. 98(4), 901-906.

11. Singh, A. (1978). Introduction: interconversion of singlet oxygenand related species. Photochem. Photobiol. 28, 429-433.

12. Khan, A. (1978). Activated oxygen singlet molecular oxygen andsuperoxide anion. Photochem. Photobiol. 28, 615-627.

EXAMPLE 14 Use of Recombinant SOD in Treating Spinal Cord Ischemia

In a new indication, demonstrated by the following example, humansuperoxide dismutase has been shown to reduce reperfusion injuryfollowing spinal ischemia. The human superoxide dismutase utilized inthis example was produced by the host-vector system pSODβ₁ Tll in E.coli A1645 described in Example 3, and grown and purified under theconditions described in Example 9.

Anesthetized dogs were connected to the Nicolet Compact 4 evokedpotential system, and we obtained the baseline SEP (Somatosensory EvokedPotentials) by applying 250 stimuli consecutively at the rate of 4.7stimuli per second to the posterior tibial nerve. The evoked potentialof 250 stimuli were recorded from 2 electrodes over the Fpz and Cz (twospecific points over the scalp), averaged by signal averager to reducethe signal to noise ratio, and the SEP was displayed on a screen.

A left thoracotomy was then performed, the descending aorta just distalto the left subclavian artery was dissected and isolated in preparationfor the application of the crossclamp. A purse string was insertedproximal to the proposed site of the aortic crossclamp and a size 20gauge cannula inserted for monitoring the proximal aortic pressure andinfusion of the medication as in the experimental group. A size 14 gaugecannula was inserted into the right femoral artery for BP monitoring andremoval of blood to control BP after the aortic crossclamp is applied.Serial blood gases were taken and the respirator was adjusted tomaintain the blood gases within normal limits.

The aortic crossclamp was then applied just distal to the leftsubclavian artery. SEP is repeated at one minute intervals. The proximalaortic hypertension was controlled by removing blood from the femoralartery to maintain BP at 90-110 mm Hg mean. The aortic crossclamp wasmaintained for 10 more minutes after the SEP disappear. Disappearance ofthe SEP tracing signifies that the ischemia within the spinal cordproduced by the crossclamping of thoracic aorta is severe enough tocompromise the conduction of afferent impulses within the dorsal columnof the spinal cord. The crossclamp was removed 10 minutes after thedisappearance of SEP. The dogs would become hypotensive which respondedto infusion of blood, Ringer's lactate and sodium bicarbonate.

In control dogs (n=8), the animals did not receive any recombinant SOD.In the experimental animals, one group (n=8) received a bolus of 25,000units of recombinant SOD prior to removal of the crossclamp followed by5,000 units per minute for 10 minutes; the second experimental group(n=7) received 5,000 units of recombinant SOD prior to removal of thecrossclamp followed by 10,000 units per minute for 10 minutes.

Postoperatively, the neurological status of the hind limbs was accessedby Tarlov's criteria: 0=no movement in hind limbs; 1=slight movement ofhind limbs; 2=good movement of hind limbs, but unable to stand; 3=ableto stand but not normally; and 4=complete recovery. On the seventhpostoperative day, SEP were repeated and recorded for comparison to thebaseline. The animals were then sacrificed.

The results are:

Neurological status on the seventh postoperative day (POD):

Control animals (n=8) - 4 animals were grade 0 -4 animals were eithergrade 2 or 3

Experimental animals (I) - 25,000 units SOD bolus and 5,000units/minute×10 minutes. - 6 animals showed complete recovery - 2animals were in either grade 2 or 3.

Experimental animals (II) - 50,000 units SOD bolus and 10,000units/minute×10 minutes. - all 17 animals showed complete recovery.

Time taken for SEP to disappear after application of aortic crossclampvaries from 12 to 19 minutes. Since the crossclamp was maintained for 10more minutes after SEP disappear, the total crossclamp time will be morethan 20 minutes.

In the immediate postoperative period after the closure of thoracotomywound, repeat SEP were taken. In the control animals, there was no SEPtracing discernible, in contrast, the treated animals showed a return ofSEP tracing with delay in the latency of the waveform.

In summary, recombinant SOD proved to be useful in preventing neurologicinjury due to spinal cord ischemia. This method of treatment isespecially important in surgery of the aneurysms of the thoracic aorta.

EXAMPLE 15 Construction of Prototrophic Strains and Production of bGHUsing Prototrophic Hosts

Prototrophic strains of E. coli have been constructed which enable highlevel protein expression by many of the previously described plasmidseven when grown in a minimal media. The advantages of a bacterial growthprotocol using minimal media are:

a) the bacteria can be grown to a higher cell density;

b) it is easier to duplicate growth conditions as the media componentsare "simpler" and therefore of a higher quality; and

c) the media is more economical.

The preferred prototrophic strains of this invention are designatedA4200, A4255, and A4346. Strain A4255 containing the plasmid p9200 hasbeen deposited with the ATCC under Accession No. 53215, and is referredto as A4320.

Selection and Construction of the Prototrophic Strains

The following strains were screened for high growth rates on minimalmedia, and sensitivity to phageλ, λ434 and phage P1:

Strain

1. ATCC 12435

2. ATCC 23716

3. ATCC 27662

4. ATCC 25404

5. ATCC 11775

6. ATCC 25254

7. HfrC=A4134

8. W3350=A2509

9. A1645

Based on results of these studies, we focused the development of aprototrophic strain based on strains ATCC 12435 and ATCC 25404 whichwere sensitive to the above-listed phage and showed superior growthrates. Using these two strains, we constructed new strains containingthe λcI857 repressor by transducing them with P1 containing λcI857 ΔH1↑Bam H1:Tn10. Tetracycline resistant colonies were purified and cured ofP1 if necessary.

The resulting strains and their genotypes are:

A4200=ATCC 12435 (λcI852 ΔH1 ΔBam H1):Tn10

A4206=ATCC 25404 (λcI857 ΔH1 ΔBam H1):Tn10

Both strains were transformed with pHG44 yielding strains A4202 and 4207respectively.

Growth and Induction

Strains A4202 and A4207 were grown and induced under the followingconditions and assayed for production of bovine growth hormone analog.

Media

    ______________________________________                                        Component             Concentration                                           ______________________________________                                        KH.sub.2 PO.sub.4     13.6   gm/liter                                         (NH.sub.4).sub.2 SO.sub.4                                                                           2      gm/liter                                         MgSO.sub.4.7H.sub.2 O 0.2    gm/liter                                         CaCl.sub.2            0.01   gm/liter                                         FeSO.sub.4.7H.sub.2 O 0.5    g/liter                                          pH 7.4                                                                        ______________________________________                                        Supplements                                                                   ______________________________________                                        Glucose 20% solution  25     ml/liter                                         Ampicillin 20 mg/ml solution                                                                        1      ml/liter                                         B1 0.3% solution      1      ml/liter                                         Biotin 0.3% solution  1      ml/liter                                         ______________________________________                                    

Both A4202 and A4207 grow well in minimal media; however, only A4202expresses significant levels of the bGH analog. A4202 expresses the bGHanalog at roughly the same level as pHG44 grown in rich media.

Elimination of Tetracycline Resistance From A4200

In order to utilize the prototrophic strain A4200 with plasmids carryingonly a tetracycline resistance marker, we constructed a strain cured ofthe Tn10 marker. Strain A4200 was streaked on MacConky galactose plates,gal⁺ revertants were selected and tested for sensitivity to tetracyclineand immunity to lambda phage. This strain was designated A4255.

Construction of Biotin Independent Prototrophic Strains

All of the above prototrophic strains contain the lambda cI857 delta H1delta Bam H1 defective prophage. The delta H1 deletion extends to thebio uvr B region, removing the biotin biosynthetic operons. Thus thestrains require the addition of biotin to the growth media.

To eliminate the biotin requirement of strains derived from A4200 andA4255, we have introduced an F' episome from strain A89 into thesestrains.

Strain A89 carries an F' gal plasmid. We have demonstrated that this F'plasmid carries all the genes necessary for the endogenous synthesis ofbiotin. Several properties make this plasmid a convenient source of thebio operons:

1. F' gal is a unit copy plasmid.

2. The plasmid is extremely stable in E. coli.

3. F' plasmids are compatible with colE1 plasmid, on which ourexpression vectors are based.

4. F' gal is a conjugative plasmid. It can, therefore, be easilytransferred from cell to cell.

5. One can easily screen for biotin independent strain.

Strain A4202 was conjugated for 30 minutes with A89, and biotinindependent colonies were selected. The resulting strain A4346 yieldshigh levels of bovine growth hormone analog following induction inminimal glucose medium in the absence of biotin. No other growth factoris required.

A4346 can be cured of pHG44 using standard methods known to thoseskilled in the art. The resulting prototrophic host strain can betransformed by all the plasmids described in this application.

Minimal Media

The minimal media standardly used for production purposes with theprototrophic strains was:

    ______________________________________                                                     For        For                                                                Fermenters Shake Flasks                                          ______________________________________                                        K.sub.2 HPO.sub.4                                                                            6      g/l       6    g/l                                      KH.sub.2 PO.sub.4                                                                            4      g/l       4    g/l                                      NH.sub.4 Cl    1      g/l       1    g/l                                      MgSO.sub.4.7H.sub.2 O                                                                        3      g/l       0.2  g/l                                      10% FeNH.sub.4 0.3    ml/l      0.1  ml/l                                     Citrate                                                                       Trace Elements 3      ml/l      1    ml/l                                     Solution                                                                      Antifoam,      0.5    ml/l                                                    Silicone                                                                      Autoclave.                                                                    ______________________________________                                    

Ampicillin (100 mg/l) or tetracycline (12.5 mg/l) may be added to themedia depending on whether the strain is ampicillin or tetracyclineresistant, respectively.

50% glucose was autoclaved separately and added to 20 gm/l. 50% glucosewas fed during the fermentation at a rate of approximately 1.08gm/glucose per O.D. unit for strains without the F' episome, or at arate of 1.8 gm/glucose per 2.0 unit for strain A4346. The pH wascontrolled by feeding 25% NH₄. Antifoam was added as needed. Biotin wasadded at 15 mg/l for strains based on A4200, A4255 and A4206.

The trace elements solution contains (Biotechnol. Bioeng. 16:933-941(1974)):

    ______________________________________                                                              g/l                                                     ______________________________________                                        H.sub.3 BO.sub.3        0.57                                                  CuCl.sub.2 (CuSO.sub.4.5H.sub.2 O)                                                                    1.0 (0.04)                                            CaCl.sub.2.2H.sub.2 O   1.0                                                   MnSO.sub.4.4H.sub.2 O   0.81                                                  Na.sub.2 MoO.sub.4.2H.sub.2 O                                                                         2.0                                                   CaCl.sub.2.6H.sub.2 O   2.0                                                   ZnCl.sub.2.4H.sub.2 O (ZnSO.sub.4.7H.sub.2 O)                                                         2.0 (2.78)                                            Concentrated HCl        100 ml                                                ______________________________________                                    

The compounds in parenthesis are alternate compounds which may be usedin place of the compounds preceding them. The parenthesized amountsrefer to appropriate amounts of such alternative compounds. Many of thepreviously described plasmids have been introduced into the prototrophsA4200 and A4255. A partial list of these strains is described in thetable below:

    ______________________________________                                        Strain Designation      Host Strain/Plasmid                                   ______________________________________                                        A4202         =         A4200/pHG44                                           A4256         =         A4255/pHG44                                           A4320         =         A4255/p9200                                           Z1803         =         A4255/pSODβ.sub.1 T11                            A4500         =         A4255/pTV 194                                         A4346         =         A4200/pHG44, F'Gal                                    ______________________________________                                    

EXAMPLE 16 Construction of Lytic Hosts and Production of bGH Using TheseLyric Hosts 1. Construction of Strains 4048 and A3111

The strain W3350 has been used extensively for growing phage lambda(see, for example, Oppenheim and Salomon (1970), 41 Virology, 151-159).Strain A2509, a prototrophic derivative of W3350, was transduced byPlcIts grown on A1637, and also transduced with a λCI857 ΔH1ΔBam H1defective prophage carrying the Tn10 marker. The resulting strain A4048was selected as a tetracycline resistant clone carrying the defectiveprophage λcI857ΔH1ΔBam H1. This strain also carried a PlcIts plasmid.This strain was transformed by pHG44 to yield strain A3111.

Similarly, strain A4085 was also constructed from A2509 by the insertionof λcI857ΔH1ΔBam H1, the removal of the Tn10 transposon and the curingof the PlcIts plasmid prior to transformation with pHG44. Strain A4085carries the defective prophage λcI857ΔH1ΔBam H1 and serves as a controlfor measuring bGH production and the autolysis affected without thepresence of the P1 plasmid. Strain A3111 has been deposited in the ATCCunder Accession No. 53217.

2. Synthesis of Bovine Growth Hormone (bGH).

Stock Cultures:

Stock cultures of strain A3111 (pHG44 in A4048 cells) were grownovernight at 30° C. in LB medium containing 50 μg/ml ampicillin (Amp).the cultures were diluted two-fold with 50% glycerol and stored at -20°C.

Inoculum:

The inoculum was obtained from a single colony of A3111 grown on an LBagar plate containing 100 μg/ml Amp. The LB plates, in turn, were spreadwith material taken from the stock cultures.

Sterile 3 ml LB medium containing 50 μg/ml Amp was inoculated with asingle colony of A3111 and grown for 18 hours at 30° C. in a shakerbath.

Production:

Production of bGH was carried out in BHI medium (37 μg/l brain heartinfustion (Difco)) containing 50 μg/ml Amp. The inoculum was diluted1:100 into a flask containing fresh BHI+50 μg/ml Amp, and grown in ashaker bath at 30° C. until the cell concentration reached about 4×10⁸cells/ml (OD.₆₀₀ =0.5).

For the induction of bGH production, the flask was transferred to ashaker bath set at 42° C. Cell samples were taken at time 0 and at 90minutes after the beginning of induction.

Analysis of bGH Production:

bGH production was analyzed on a 10-26% gradient acrylamide gel. Thecell samples were spun in a microfuge, the supernatant was removed, andthe pellets were dissolved in sample buffers (2% SDS, 50 mM Tris pH 7.0,3% sucrose, 5% β-Mercaptoethanol) and loaded on the gel. Afterelectrophoresis at 200 volts for 2-1/2 hours, the gels were stained withCoomassie blue and the amount of bGH was determined by scanning with agel scanner.

After 90 minutes of induction at 42° C., bGH comprises about 20% of thetotal protein of A3111.

3. Autolysis

Strain A3111 carries. pHG44, the defective prophage λcI857ΔH1 ΔBam H1,and a stable PlcIts plasmid. After prolonged induction at 42° C., thecells will start to lyse due to the production of endolysin directed byP1. Complete lysis of the culture was achieved after 2.5-3 hrs.

Test of Controlled Autolysis:

5 ml of A3111 culture were taken before and after induction at 42° C.(90 min.) and spun down in a Sorvall centrifuge at 7000 rpm for 7minutes. The supernatants were removed and the pellets were frozen at-20° C. overnight. The pellets were then resuspended in 0.5 ml of T.E.buffer (10 mM Tris pH 8.0, 1 mM EDTA) and 0.1 ml was diluted 1:10 withT.E. and the OD.₆₀₀ determined.

As a control for this experiment we used strain A4085 which, similarlyto A3111, contains pHG44 and the defective prophage λcI857ΔH1ΔBam H1 butdoes not carry the PlcIts plasmid.

The results of such an experiment appear in Table III which shows thatcells containing the PlcIts plasmid (A3111) lyse immediately uponthawing. Inspection of the thawed mixture revealed that over 95% of thecells were lysed following this treatment.

                  TABLE III                                                       ______________________________________                                                  OD..sub.600                                                                         Before                                                        Strain PlcIts   Freezing    After Thawing                                                                           % Loss                                  ______________________________________                                        A4085  -        0.980       0.851     14                                      A3111  +        0.543       0.08      86                                      ______________________________________                                    

The lysis procedure simplifies the extraction of bGH from the inducedcells without affecting bGH production.

EXAMPLE 17 Toxicological Evaluation of hCuZnSOD

Toxicology studies of the hSOD prepared in Example 9 were carried out onthe Macace Fasicularis (Cynomolgus) monkey.

0.2 g/kg Study

Two males and two females were dosed with hSOD at 0.2 g/kg, The studywas performed by intravenous bol us injection in physiological saline ata concentration of 150 mg/ml. The indices of safety evaluation wereweight change, hematology, serum chemistry, urinalysis, dailyobservation, renal biopsy (pre-dose and 72 hr), renal immunochemistry(IgM and IgG) and pathological evaluation. No essential test resultswere reported that could be attributed to test article administration inthis study. More specifically, no evidence of pathology in the renalcortex at 72 hr or at sacrifice (144 hr) was observed. The serumchemistry indices of renal function remained normal throughout thestudy. The right or contralateral kidney which was evaluated at necropsygave no evidence of test article related toxicity either by gross orhistopathological examination. Immunochemistry or immunohistopathologyfor IgM or IgG was unremarkable as well.

0.3 g/kg Study

Two males and two females were dosed with hSOD prepared as described inExample 9. The dose injected in this study was 0.3 g/kg by intravenousbolus injection dissolved in physiological saline.

The parameters measured for both general health effects of hSOD and itsnephrotoxicity included daily observations, serum and urine analysis,renal biopsy, renal immunohistopathology and histopathology. No evidenceof transitory renal pathology or immunological reaction was observed.However, slight increase in serum BUN (blood-urea-nitrogen) levels wereobserved, which returned to normal by sacrifice at 144 hr, creatininelevels showed mild elevation within normal range. In summary, there wasno evidence of immunohistopathology in the kidney (or elsewhere) and noessential test results could be attributed to test articleadministration in this study.

The results of these two toxicology studies employing recombinant humanSOD analog is in marked contrast to bovine-SOD (orgotein) whose LD₅₀ inmonkeys is reported to be 10 mg/kg (Grunenthal-Sales brochure describingPEROXINORM, which is a trademark for orgotein which is bovine superoxidedismutase).

EXAMPLE 18 Activity of hCuZnSOD After Repeated Lyophylization

Human CuZnSOD as prepared according to the method described in Example 9was dissolved 1 mg/ml in H₂ O or in 10 mM NH₄ HCO₃ and divided to 1 mlsamples. Several vials were frozen and kept as a zero reference. Theother samples were lyophilized then redissolved in 1 ml of H₂ O or in 10mM NH₄ HCO₃ and lyophilized again. At each stage of lyophilizationduplicate samples from the H₂ O and from the NH₄ HCO₃ treated sampleswere withdrawn and kept at 4° C. At the end of the experimentsamples-which had either not been lyophilized or lyophilized 1, 2, 3, 4,or 5 times (designated 0, I, II, III, IV and V, respectively) wereobtained for the H₂ O and 10 mM NH₄ HCO₃ treated samples. All four setsof lyophilized Cu-Zn-hSOD were assayed for activity in triplicates. Eachof the following experiments was performed on a different day.

The experiments demonstrate that the hSOD analog produced in bacteriaretains most of its activity even after repeated lyophil izations,unlike natural hSOD which loses most of its enzymatic activity after twolyophilizations. (See U.S. Pat. No. 3,637,640.)

                  TABLE IV                                                        ______________________________________                                                                             % activity                                                                    as com-                                                                       pared to                                 Exp.                     std. std.   fresh                                    1      μ/mg   mean    dev. error  hCuZnSOD                                 ______________________________________                                        hCuZn Lyophilizations in H.sub.2 O                                            0      2771                                                                   0      3079      3036    247  142    106                                      0      3259                                                                   I      2577                                                                   I      2960      2832    221  128     99                                      I      2960                                                                   II     2381                                                                   II     2381      2465    145   84     86                                      II     2632                                                                   III    2482                                                                   III    2482      2569    151   87     90                                      III    2743                                                                   IV     2353                                                                   IV     2601      2436    143   83     85                                      IV     2353                                                                   V      2393                                                                   V      2393      2477    146   84     87                                      V      2646                                                                   fresh  2570                                                                   fresh  2840      2855    293  169    100                                      fresh  3156                                                                   ______________________________________                                                                             % activity                                                                    as com-                                                                       pared to                                 Exp.                     std. std.   fresh                                    2      μ/mg   mean    dev. error  hCuZnSOD                                 ______________________________________                                        0      3103                                                                   0      3398      3368    252  145     95                                      0      3604                                                                   I      3045                                                                   I      3225      3229    186  108     91                                      I      3418                                                                   II     3295                                                                   II     3263      3197    143   82     91                                      II     3033                                                                   III    3125                                                                   III    3308      3238     99   57     92                                      III    3281                                                                   IV     3111                                                                   IV     3081      3085     25   14     87                                      IV     3062                                                                   V      3050                                                                   V      2556      2869    272  157     81                                      V      3002                                                                   fresh  4017                                                                   fresh  3281      3529    423  244    100                                      fresh  3289                                                                   ______________________________________                                                                             % activity                                                                    as com-                                                                       pared to                                 Exp.                     std. std.   fresh                                    3      μ/mg   mean    dev. error  hCuZnSOD                                 ______________________________________                                        hCuZnSOD Lyophilizations in 10 mM NH.sub.4 HCO.sub.3                          0      2938                                                                   0      3141      3139    199  115    101                                      0      3337                                                                   I      2764                                                                   I      3130      3002    206  119     96                                      I      3111                                                                   II     2782                                                                   II     3345      3092    286  165     99                                      II     3148                                                                   III    2697                                                                   III    3035      2922    195  113     94                                      III    3035                                                                   IV     2680                                                                   IV     3224      2908    282  163     93                                      IV     2820                                                                   V      2558                                                                   V      2558      2680    211  122     86                                      V      2923                                                                   fresh  2803                                                                   fresh  3372      3110    287  166    100                                      fresh  3155                                                                   ______________________________________                                                                             % activity                                                                    as com-                                                                       pared to                                 Exp.                     std. std.   fresh                                    4      μ/mg   mean    dev. error  hCuZnSOD                                 ______________________________________                                        0      4495                                                                   0      4790      4692    170   98    120                                      0      4790                                                                   I      3842                                                                   I      3842      3842     0    0      99                                      I      3842                                                                   II     3965                                                                   II     3277      3506    397  229     90                                      II     3277                                                                   III    3210                                                                   III    3372      3485    195  113     90                                      III    3372                                                                   IV     3785                                                                   IV     3442      3671    199  115     94                                      IV     3787                                                                   V      3349                                                                   V      3558      3419    121   70     88                                      V      3349                                                                   fresh  3885                                                                   fresh  3885      3885     0    0     100                                      fresh  3885                                                                   ______________________________________                                    

EXAMPLE 19 Exclusive Expression of Non-Acetylated hCuZnSOD in E. Coli

According to the DNA sequence of the cloned human CuZn SOD in pSODβ₁T₁₁, initiation of protein synthesis could start at either one of twoATG codohs located at the 5' end of the gene (FIG. 25). Proteinsequencing studies prove that hSOD isolated from E. coli lacks anN-terminal methionine and most of it begins with an alanins residue.This result suggests that protein biosynthesis is initiated at thesecond ATG and that the methionyl residue is removed by a bacterialpeptidase. However, about 5% of the expressed enzyme containSet-Met-Alae at the amino terminus indicating that the first ATG canalso be utilized to initiate translation. The following steps wereundertaken in order to eliminate the first ATG, thus leading to theexclusive expression of non-acetylated hCuZnSOD analog having an aminoacid sequence identical to that of natural human CuZn SOD.

Construction of pΔRB

Tet^(R) expression vector, pΔRB, was generated from pSODβ₁ T₁₁ bycomplete digestion with EcoRI followed by partial cleavage with BamHIrestriction enzymes. The digested plasmid was ligated with syntheticoligomer ##STR23## resulting in p ΔRB containing the λ P_(L) promoter.(FIG. 26).

Construction of pβUN

Construction of pβUN, a general purpose expression vector, containingthe λP_(L) promoter and β-lactamase promoter and RBS is shown in FIG.27. Unique NdeI site followed by SmaI, XbaI and BglII sites wereintroduced downstream of the β-lactamase RBS for insertion of anydesired gens. It should be pointed out that 3 nucleotide changes weremade at the 3' end of the lactamase RBS - , one to eliminate the firstpossible ATG (C instead of G) and two other changes to form the NdeIsite (CA instead of AG).

Construction of pSODβMA

The entire coding region of human CuZn SOD on a NdeI-BamHI fragmentisolated from pSODα13, was inserted between the unique NdeI and BglIIsites of pβUN as depicted in FIG. 28. The resulting vector, pSODβMA,failed to express high levels of hSOD upon induction.

Construction of pSODβMAX

High level expression of authentic hSOD was achieved by a single basechange in the β-lactamase RBS region as compared to- the sequencepresent in pSODβ₁ T₁₁ 1 (FIG. 25). The 22 bp long SspiI-NdeI fragment ofpSODβMA was replaced with 3 different synthetic fragments, each having asingle base substitution which eliminates the first ATG (either C, T orA instead of G in the third position of the initiator codon). Theconstruction of the pSOD⊖MAX₁₂ and pSODβMAX₁₀ clones are described inFIGS. 29 and 30, respectively. pSODβMAX₁₂ in prototrophic E. coli hostA4255 has been deposited with the American Type Culture Collection andassigned ATCC Accession No. 67177. As shown in Table V only the G to Cand G to substitutions resulted in high level hSOD expression uponinduction.

                                      TABLE V                                     __________________________________________________________________________    SOD EXPRESSION                     % SOD                                      VECTORS    SEQUENCE AT THE 5' END  BY GEL SCAN                                __________________________________________________________________________    β.sub.1 T.sub.11                                                                     ##STR24##              6.5                                        βMA                                                                                  ##STR25##              0.1                                        βMAX.sub.10                                                                          ##STR26##              6.8                                        βMAX.sub.11                                                                          ##STR27##              0.1                                        βMAX.sub.12                                                                          ##STR28##              8.3                                        __________________________________________________________________________

What is claimed is:
 1. An analog of human superoxide dismutase whichcomprises the non-acetylated, non-glycosylated form of natural humanCuZn superoxide dismutase having the set-met amino acid sequenceattached to the amino terminus.
 2. A mixture of superoxide dismutaseanalogs comprising non-acetylated, non-glycosylated CuZn superoxidedismutase having an amino acid sequence identical to that of naturalhuman superoxide dismutase and non-acetylated, non-glycosylated analogCuZn of natural human CuZn superoxide dismutase having an amino acidsequence identical to that of natural CuZn superoxide dismutase andhaving the amino acid sequence ser-met attached to the amino terminus.3. A mixture according to claim 2, wherein the non-acetylated,non-glycosylated analog of CuZn superoxide dismutase having an aminoacid sequence identical to that of natural human CuZn superoxidedismutase ranges from about 90 to about 95% by weight of the mixture andthe non-acetylated, non-glycosylated analog of natural human CuZnsuperoxide dismutase having an amino acid sequence identical to that ofnatural CuZn superoxide dismutase and the amino acid sequence ser-metattached to the amino terminus ranges from about 5 to 10% of the mixtureby weight.
 4. A mixture according to claim 3, wherein thenon-acetylated, non-glycosylated analog of CuZn superoxide dismutasehaving an amino acid sequence identical to that of natural human CuZnsuperoxide dismutase comprises 95% of the mixture by weight and thenon-acetylated, non-glycosylated analog of CuZn superoxide dismutasehaving an amino acid sequence identical to that of natural human CuZnsuperoxide dismutase and having the ser-met amino acid sequence at theamino terminus comprises about 5% of the mixture by weight.
 5. A mixtureaccording to claim 3, wherein the non-acetylated, non-glycosylatedanalog of CuZn superoxide dismutase having animo acid sequence identicalto that of natural human CuZn superoxide dismutase comprises more thanabout 93% of the mixture by weight and the non-acetylated,non-glycosylated analog of CuZn superoxide dismutase having an aminoacid sequence identical to that of natural human CuZn superoxidedismutase and having the ser-met amino acid sequence at the aminoterminus comprises less than about 7% of the mixture by weight.
 6. Amethod of catalyzing the reaction

    20.sub.2-+2 H+→H.sub.2 O.sub.2 +O.sub.2

which comprises contacting the reactants under suitable conditions withan analog of human CuZn superoxide dismutase in accordance with claim 1.7. A method of catalyzing the reaction

    20.sub.2-+2 H+→H.sub.2 O.sub.2 +O.sub.2

which comprises contacting the reactancts under suitable conditions withsuperoxide dismutase analogs in accordance with a mixture of CuZn claim2.